Spray freeze-dried compositions

ABSTRACT

A process for producing a powder comprises spray freeze-drying an aqueous solution or suspension comprising a pharmaceutical agent, said solution or suspension having a solids content of 20% by weight or more. The spray freeze-dried powder may be administered to a subject via a needleless syringe.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to U.S. provisional patent application Ser.No. 60/296,939, filed 8 Jun. 2001, from which application priority isclaimed pursuant to 35 U.S.C. §119(e)(1) and which application isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to processes for producing pharmaceuticalcompositions which are suitable for transdermal particle delivery from aneedleless syringe system.

BACKGROUND TO THE INVENTION

The ability to deliver pharmaceutical agents into and through skinsurfaces (transdermal delivery) provides many advantages over oral orparenteral delivery techniques. In particular, transdermal deliveryprovides a safe, convenient and noninvasive alternative to traditionaladministration systems, conveniently avoiding the major problemsassociated with oral delivery (e.g. variable rates of absorption andmetabolism, gastrointestinal irritation and/or bitter or unpleasant drugtastes) or parenteral delivery (e.g. needle pain, the risk ofintroducing infection to: treated individuals, the risk of contaminationor infection of health care workers caused by accidental needle-sticksand the disposal of used needles).

However, despite its clear advantages, transdermal delivery presents anumber of its own inherent logistical problems. Passive delivery throughintact skin necessarily entails the transport of molecules through anumber of structurally different tissues, including the stratum corneum,the viable epidermis, the papillary dermis and the capillary walls inorder for the drug to gain entry into the blood or lymph system.Transdermal delivery systems must therefore be able to overcome thevarious resistances presented by each type of tissue.

In light of the above, a number of alternatives to passive transdermaldelivery have been developed. These alternatives include the use of skinpenetration enhancing agents, or “permeation enhancers,” to increaseskin permeability, as well as non-chemical modes such as the use ofiontophoresis, electroporation or ultrasound. However, these alternativetechniques often give rise to their own unique side effects such as skinirritation or sensitization. Thus, the spectrum of agents that can besafely and effectively administered using traditional transdermaldelivery methods has remained limited.

More recently, a novel transdermal drug delivery system that entails theuse of a needleless syringe to fire powders (i.e., solid drug-containingparticles) in controlled doses into and through intact skin has beendescribed. In particular, commonly owned U.S. Pat. No. 5,630,796 toBellhouse et al. describes a needleless syringe that deliverspharmaceutical particles entrained in a supersonic gas flow. Theneedleless syringe is used for transdermal delivery of powdered drugcompounds and compositions, for delivery of genetic material into livingcells (e.g., gene therapy) and for the delivery of biopharmaceuticals toskin, muscle, blood or lymph. The needleless syringe can also be used inconjunction with surgery to deliver drugs and biologics to organsurfaces, solid tumors and/or to surgical cavities (e.g., tumor beds orcavities after tumor resection). In theory, practically anypharmaceutical agent that can be prepared in a substantially solid,particulate form can be safely and easily delivered using such devices.

To enable powdered drug compositions to be effectively administered viathis new needleless syringe technique, the powders should have certainphysical characteristics. In particular, the size of the particles whichform the powders should be controllable, preferably with a narrow sizedistribution. Further, the particle density should be high, theparticles should be free-flowing under a dry environment and theirmoisture content should be low. Additional properties of the particleswhich are desired include a spherical shape and a smooth surface. Eachof these properties is important to provide good skin penetration whilstavoiding damage to the particles themselves under the forces requiredfor delivery via needleless syringe.

One of the most important factors in determining the physicalcharacteristics of the powders is the particular manner by which theyare produced. Various spray freeze-drying techniques have previouslybeen described for, for example, the preparation of powders for aerosoldelivery and microspheres for conventional drug delivery. In theseapplications, particles are desired to be light and porous, propertieswhich are inherent in powders produced by previously described sprayfreeze-drying techniques. Such light and porous particles are notsuitable for use in needleless syringe devices.

Maa et al. (1999) Pharmaceuticals Research 16(2) describe the physicalcharacteristics of spray-freeze-dried particles and their performance asaerosols. The spray freeze-drying process is said to render highlyporous particles with a large specific surface area. Maa estimated thatthe particle density of spray freeze-dried particles is typicallyapproximately one ninth of that of equivalent particles dried byspray-drying.

U.S. Pat. No. 5,019,400 describes a process for preparing microspheresusing very cold temperatures to freeze polymer-biologically active agentmixtures into polymeric microspheres with retention of biologicalactivity and material. Polymer is dissolved in a solvent together withan active agent that can be either dissolved in the solvent or dispersedin the solvent in the form of microparticles. The polymer/active agentmixture is atomised into a vessel containing a liquid non-solvent, aloneor frozen and overlayed with a liquified gas, at a temperature below thefreezing point of the polymer/active agent solution.

When the combination with the liquified gas is used, the atomiseddroplets freeze into microspheres upon contacting the cold liquifiedgas, then sink onto the frozen non-solvent layer. The frozen non-solventis then thawed. As the non-solvent thaws, the microspheres which arestill frozen sink into the liquid non-solvent. The solvent in themicrospheres then thaws and is slowly extracted into the non-solvent,resulting in hardened microspheres containing active agent either as ahomogeneous mixture of the polymer and the active agent or as aheterogeneous two phase system of discrete zones of polymer and activeagent.

If a cold solvent is used alone, the atomized droplets freeze uponcontacting the solvent, and sink to the bottom of the vessel. As thenon-solvent for the polymer is warmed, the solvent in the microspheresthaws and is extracted into the non-solvent, resulting in hardenedmicrospheres.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that spray freeze-drying asolution or suspension having a high solids content in the solventproduces particles which are quite dense and which perform well inneedleless syringe devices. The use of a high solids content startingmaterial minimises the pore formation which occurs during the dryingstep. Sublimation of frozen solvent away from the particles duringdrying is the typical cause of pore formation. Maximising the solidscontent in the solution or suspension, and therefore in the frozenparticles, reduces the number and size of pores which form on drying,thus providing denser particles.

The inventors have also found that the use of particular excipients mayaid the formation of dense particles. Appropriate excipient compositionsallow particles to collapse and density during freeze-drying. It is alsothought that selecting specific excipient compositions may assistparticle formation by increasing the solubility of certain guestsubstances (such as peptides and proteins) in the solvent system. Thisfurther aids in maximising the solids content of the solution orsuspension.

The present invention therefore enables the spray freeze-drying process,with its attendant advantages, to be adapted to needless syringerequirements. The particles of the invention have a well-defined sizeand a high density, together with other mechanical properties whichcollectively are suitable for transdermal delivery via a needlelesssyringe. The present spray freeze-drying process is a simple techniquewhich is highly suitable for scaling-up to commercial production levels.In addition, it has, as yet, been found to be entirely formulationindependent. The technique can therefore be applied to almost anypharmaceutical formulation, a factor which further adds to thecommercial viability of the present invention.

Accordingly, the present invention provides a process for thepreparation of a powder, which process comprises the step of sprayfreeze-drying an aqueous solution or suspension comprising apharmaceutical agent, said solution or suspension having a solidscontent of 20% by weight or more.

The invention further provides:

a dosage receptacle for a needleless syringe, said receptacle containingan effective amount of a powder prepared by the process of theinvention;

a needleless syringe which is loaded with a powder prepared by theprocess of the invention;

a vaccine composition comprising a pharmaceutically acceptable carrieror diluent and a powder prepared by the process of the invention;

a method of vaccinating a subject, which method comprises administeringto the said subject an effective amount of a powder prepared by theprocess of the invention; and

a powder which is prepared by the process of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-d are optical micrographs (at ×100 magnification) of selectedparticle formulations assessed in the study described in Example 1 atsubpart 1.5.2.

FIG. 1 a shows particles from Formulation 138-20-4A, prepared by sprayfreeze-drying (SFD) 20 μg of the HBsAg antigen. FIG. 1 b shows particlesfrom Formulation 138-16-1C, prepared by SFD and then sieving to obtain a38-53 μm particle fraction formed from 20 μg of HBsAg antigen and 50 μgof alum adjuvant. FIG. 1 c shows particles from Formulation 138-20-4B,prepared by SFD and then compressing, grinding and sieving (C/G/S) toobtain a 38-53 μm particle fraction formed from 20 μg of HBsAg antigen.FIG. 1 d shows particles from Formulation 138-20-5C, prepared by SFD andthen C/G/S to obtain a 38-53 μm particle fraction formed from 20 μg ofHBsAg antigen and 50 μg of alum adjuvant.

FIGS. 2 a-2 d are SEM micrographs of selected particle formulationsassessed in the study described in Example 1 at subpart 1.5.3. FIG. 2 ashows particles from Formulation 138-20-4A, prepared by SFD 20 μg of theHBsAg antigen. FIG. 2 b shows particles from Formulation 138-16-1C,prepared by SFD and then sieving to obtain a 38-53 μm particle fractionformed from 20 μg of HBsAg antigen and 50 μg of alum adjuvant. FIG. 2 cshows particles from Formulation 138-20-4B, prepared by SFD and thenC/G/S to obtain a 38-53 μm particle fraction formed from 20 μg of HBsAgantigen. FIG. 2 d shows particles from Formulation 138-20-5C, preparedby SFD and then C/G/S to obtain a 38-53 μm particle fraction formed from20 μg of HBsAg antigen and 50 μg of alum adjuvant.

FIGS. 3 a and 3 b are optical images of liquid suspensions of twoalum-containing formulations assessed in the study described in Example1 at subpart 1.5.7. FIG. 3 a is an optical image of the reconstitutedformulation 138-16-1 prepared by SFD 20 μg of the HBsAg antigen. FIG. 3b is an optical image of the reconstituted formulation 138-20-5Cprepared by SFD and then C/G/S to obtain a 38-53 μm particle fractionformed from 20 μg of HBsAg antigen and 50 μg of alum adjuvant.

FIGS. 4 a and 4 b are photos of the non-reducing SDS-PAGE gel (FIG. 4 a)and reducing SDS-PAGE gel (FIG. 4 b) showing the results of the studydescribed in Example 1 at subpart 1.5.8.

FIG. 5 is a schematic of the Spray Freeze-Drying (SFD) process describedin Example 2 at subpart 2.3.1.

FIGS. 6 a-6 d are optical micrographs (at ×100 magnification) ofselected particle formulations assessed in the study described inExample 2 at subpart 2.5.1. FIG. 6 a shows particles from Formulation156-16-1 prepared by SFD a composition containing 10% BSA, 45%trehalose, 27% mannitol, 18% PVP (K17) and 0.1% Pluronic F68. FIG. 6 bshows particles from Formulation 156-16-2 prepared by SFD a compositioncontaining 10% BSA, 44.9% trehalose, 26.9% mannitol, 18% PVP (K17), 0.1%methionine, and 0.1% Pluronic F68. FIG. 6 c shows particles fromFormulation 156-16-3 prepared by SFD a composition containing 10% BSA,26.9% trehalose, 26.9% mannitol, 35.9% PVP (K17), 0.1% methionine, and0.1% Pluronic F68. FIG. 6 e shows particles from Formulation 156-16-4prepared by SFD a composition containing 10% BSA, 26.9% trehalose, 26.9%mannitol, 35.9% PVP (K17), 0.1% methionine, and 0.1% Pluronic F68.

FIGS. 7 a-7 f are optical micrographs (at ×100 magnification) ofselected particle formulations assessed in the study described inExample 2 at subpart 2.5.2.

FIG. 7 a shows particles from Formulation 156-35-1 prepared by SFD acomposition containing 10% BSA, 36% raffinose, 27% trehalose, and 27%mannitol. FIG. 7 b shows particles from Formulation 156-35-2 prepared bySFD a composition containing 10% BSA, 36% raffinose, 36% mannitol, and18% PVP (K17). FIG. 7 c shows particles from Formulation 156-35-3prepared by SFD a composition containing 10% BSA, 40% raffinose and 30%mannitol. FIG. 7 d shows particles from Formulation 156-42-1 prepared bySFD a composition containing 10% BSA, 36% raffinose, 27% trehalose, and27% mannitol. FIG. 7 e shows particles from Formulation 156-42-2prepared by SFD a composition containing 10% BSA, 27% raffinose, 27%mannitol, 18% glycine and 18% trehalose. FIG. 7 f shows particles fromFormulation 156-42-4 prepared by SFD a composition containing 10% BSA,27% raffinose, 27% sucrose, and 36% mannitol

FIGS. 8 a-8 f are optical micrographs (at ×100 magnification) ofselected particle formulations assessed in the study described inExample 2 at subpart 2.5.3. FIG. 8 a shows particles from Formulation156-35-4 prepared by SFD a composition containing 10% BSA, 27%trehalose, 27% mannitol and 36% dextran (10 kDa). FIG. 8 b showsparticles from Formulation 156-42-3-1 prepared by SFD a compositioncontaining 10% BSA, 27% trehalose, 27% mannitol and 36% dextran (10kDa). FIG. 8 c shows particles from Formulation 156-42-3-2 prepared bySFD composition containing 10% BSA, 27% trehalose, 27% mannitol and 36%dextran (10 kDa). FIG. 8 e shows particles from Formulation 156-61-1prepared by SFD a composition containing 10% BSA, 27% trehalose, 27%mannitol and 36% dextran (10 kDa). FIG. 8 f shows particles fromFormulation 156-65-1 prepared by SFD a composition containing 10% BSA,36% trehalose, 18% mannitol, 18% arginine glutamate, and 18% dextran (10kDa).

FIGS. 9 a-9 f are optical micrographs (at ×100 magnification) ofselected particle formulations assessed in the study described inExample 2 at subpart 2.5.4. FIG. 9 a shows particles from Formulation156-57-1 prepared by SFD a composition containing 10% BSA, 36%trehalose, 36% mannitol, and 18% alanine. FIG. 9 b shows particles fromFormulation 156-57-2 prepared by SFD a composition containing 10% BSA,27% trehalose, 27% mannitol, and 36% arginine glutamate. FIG. 9 c showsparticles from Formulation 156-65-2 prepared by SFD a compositioncontaining 10% BSA, 36% trehalose, 18% mannitol, and 36% arganineglutamate. FIG. 9 d shows particles from Formulation 156-71-1 preparedby SFD a composition containing 10% BSA, 36% trehalose, 18% mannitol,and 36% arganine glutamate. FIG. 9 d shows particles from Formulation156-76-1 prepared by SFD a composition containing 10% BSA, 35.9%trehalose, 18% mannitol, 35.9% arginine glutamate, 0.1% Pluronic F168,and 0.1% methionine. FIG. 9 f shows particles from Formulation 156-76-2prepared by SFD a composition containing 10% BSA, 26.9% trehalose, 26.9%mannitol, 35.9% arginine glutamate, 0.1% Pluronic F168, and 0.1%methionine.

FIGS. 10 a-10 c are optical micrographs (at ×100 magnification) ofselected particle formulations assessed in the study described inExample 2 at subpart 2.5.5. FIG. 10 a shows particles from Formulation156-80-1 prepared by SFD a composition containing 10% BSA, 27%trehalose, 27% mannitol, and 36% arginine aspartate. FIG. 10 b showsparticles from Formulation 156-80-2 prepared by SFD a compositioncontaining 10% BSA, 5% Pluronic F168, 59.5% trehalose, and 25.5%mannitol. FIG. 10 c shows particles from Formulation 156-80-3 preparedby SFD a composition containing 10% BSA, 35.9% trehalose, 18% mannitol,35.9% arginine glutamate, 0.1% methionine and 0.1% Tween 80.

FIGS. 11 a-11 d are optical micrographs of selected particleformulations assessed in the study described in Example 3 at subpart3.4. FIG. 11 a shows the Adju-Phos adjuvant (2 w/v % placebo AlPO₄) gelafter freezing at −20° C. and thawing under ambient conditions. FIG. 11b shows the Adju-Phos gel after spray-freezing and then thawing. FIG. 11c shows the Alhydrogel adjuvant (2 w/v % placebo Al(OH)₃) gel afterfreezing at −20° C. and thawing at ambient conditions. FIG. 11 d showsthe Alhydrogel gel after spray-freezing and then thawing.

FIGS. 12 a and 12 b depict the particle size analysis described inExample 3, subpart 3.5.1. FIG. 12 a shows the particle size analyses byAccuSizer for: (a) a starting alum-adsorbed HBsAg gel, depicted by the(♦) curve on the graph; (b) a “high Alum SFD” composition formed fromAlum hydroxide (3.0 w/v %), mannitol (1.9 w/v %), glycine (0.5 w/v %),and dextran (0.61 w/v %), depicted by the (♦) curve on the graph; and(c) a “low Alum. SFD” composition formed from Alum hydroxide (0.6 w/v%), mannitol (2.8 w/v %), glycine (1.2 w/v %), and dextran (0.58 w/v %),depicted by the (*) curve on the graph. FIG. 12 b shows the particlesize analyses by AccuSizer for: (a) a starting alum-adsorbed HBsAg gel,depicted by the (♦) curve on the graph; and a freeze dried, C/G/Sprocessed composition formed from Alum hydroxide (3.0 w/v %), mannitol(1.9 w/v %), glycine (0.5 w/v %), and dextran (0.61 w/v %), depicted bythe (*) curve on the graph.

FIG. 13 shows the ELISA results obtained in Example 3, subpart 3.5.1,reported as anti-HBsAg antibody responses elicited in the immunizedanimals receiving “FD” (SFD composition formed from Alum hydroxide (3.0w/v %), mannitol (1.9 w/v %), glycine (0.5 w/v %), and dextran (0.61 w/v%)); “FD<20 μm” (SFD composition formed from Alum hydroxide (3.0 w/v %),mannitol (1.9 w/v %), glycine (0.5 w/v %), and dextran (0.61 w/v %),particle size 20 μm fraction); “FD 38-53 μm” (SFD composition formedfrom Alum hydroxide (3.0 w/v %), mannitol (1.9 w/v %), glycine (0.5 w/v%), and dextran (0.61 w/v %), particle size 38-53 μm fraction); “FD53-75 μm” (SFD composition formed from Alum hydroxide (3.0 w/v %),mannitol (1.9 w/v %), glycine (0.5 w/v %), and dextran (0.61 w/v %),particle size 53-78 μm fraction); “SFD 3% Alum” (SFD composition formedfrom Alum hydroxide (3.0 w/v %), mannitol (1.9 w/v %), glycine (0.5 w/v%), and dextran (0.61 w/v %)); “SFD 0.6% Alum” (SFD composition formedfrom Alum hydroxide (0.6 w/v %), mannitol (2.8 w/v %), glycine (1.2 w/v%), and dextran (0.58 w/v %)); and “untreated” the untreated controlanimals.

FIGS. 14 a-14 b show the ELISA results obtained in Example 3, subpart3.5.2, reported as anti-dT (FIG. 14 a) and anti-tT (FIG. 14 b) antibodyresponses elicited in the immunized animals receiving either: (a) the“SFD” composition formed from alum phosphate (1.5 w/v %), trehalose.(1.5 w/v %), glycine (0.4 w.v %), and dextran (0.6 w/v %), delivered byepidermal powder injection (“EPI”); (b) the “SD” composition formed fromalum phosphate (5 w/v %) and trehalose (5 w/v %), delivered by EPI; or(c) the “untreated” composition which was a liquid DT vaccinecomposition administered by intramuscular (IM) injection.

FIGS. 15 a-15 e are digital light microscope images of H&E stainedsections from the histological skin samples taken in the study describedin Example 4, subpart 4.4.1, and showing histological changes in theimmunization sites. FIG. 15 a shows normal skin (20×); FIG. 15 b showsthe site of EPI administration (24×); FIG. 15 c shows the site of EPIadministration (105×); FIG. 15 d shows the site of intradermal (ID)injection (20×) and FIG. 15 e shows the site of ID injection (105×).

FIGS. 16 a and 16 b are scanning electron micrographs (SEMs) of SFD alumpowder formulations described in Example 4 at subpart 4.5.1. Theformulations were formed from compositions containing trehalose (30%),mannitol (30%), dextran (40%) at 35 w/w % of total solid content. FIG.16 a shows an aluminium hydroxide composition, and FIG. 16 b shows analuminium phosphate composition.

FIGS. 17 a-17 d are optical micrographs assessing alum coagulation inselected particle formulations as described in Example 4 at subpart4.4.1. FIG. 17 a shows a reconstituted freeze dried (FD) alum hydroxidecomposition formulated with trehalose (30%), mannitol (30%), dextran(40%) at 35 w/w % of total solid content.

FIG. 17 b shows a reconstituted SFD alum hydroxide compositionformulated with trehalose (30%), mannitol (30%), dextran (40%) at 35 w/w% of total solid content. FIG. 17 c shows a reconstituted FD alumphosphate composition formulated with trehalose (30%), mannitol (30%),dextran (40%) at 35 w/w % of total solid content. FIG. 17 d shows areconstituted SFD alum phosphate composition formulated with trehalose(30%), mannitol (30%), dextran (40%) at 35 w/w % of total solid content

FIGS. 18 a-18 b are optical micrographs assessing alum coagulation inselected particle formulations as described in Example 4 at subpart4.4.3. FIG. 18 a shows a reconstituted SFD alum phosphate compositionformulated with trehalose (30%), mannitol (30%), dextran (40%) andpolysorbate 80 at 0.5 w/w % of total solid content, where the startingtotal solid content was 35 w/w %. FIG. 18 b shows a reconstituted SFDalum phosphate composition formulated with trehalose (30%), mannitol(30%), dextran (40%) and polysorbate 80 at 0.5 w/w % of total solidcontent, where the starting total solid content was 40 w/w %. FIG. 18 cshows a reconstituted SFD alum phosphate composition formulated withtrehalose (30%), mannitol (30%), dextran (40%) and polysorbate 80 at 0.5w/w % of total solid content, where the starting total solid content was30 w/w %. FIG. 18 d shows a reconstituted SFD alum phosphate compositionformulated with trehalose (30%), mannitol (30%), dextran (40%) andpolysorbate 80 at 0.5 w/w % of total solid content, where the startingtotal solid content was 25 w/w %.

FIGS. 19 a and 19 b show the ELISA results obtained in Example 4,subpart 4.6.1, reported as geometric mean anti-HBsAg IgG antibody titersof animals vaccinated with HBsAg vaccine compositions. FIG. 19 a showstiters from animals receiving SFD compositions delivered by EPI(“SFD/EPI”), where the compositions were either a SFD formulationcontaining 1 μg HBsAg adsorbed to 25 μg aluminium hydroxide (“Al(OH)₃”)or a SFD formulation containing 1 μg HBsAg adsorbed to 25 μg aluminiumphosphate (“AlPO₄”); animals receiving SFD compositions that werereconstituted to liquid form and delivered via IM injection(“SFD/reconstituted/IM”), where the compositions were either a SFDformulation containing 1 μg HBsAg adsorbed to 25 μg aluminium hydroxide(“Al(OH)₃”) or a SFD formulation containing 1 μg HBsAg adsorbed to 25 μgaluminium phosphate (“AlPO₄”); or a control receiving the commercialHepatitis B vaccine composition containing 1 μg HBsAg adsorbed to 25 μgAl(OH)₃. Titers are reported from week 4, 6 and 9 sera. FIG. 19 b showstiters from animals receiving either a reconstituted SFD vaccineformulation containing 2 μg HBsAg adsorbed with 2.5 μg AlPO₄(“SFD/reconstituted”); or a liquid injection of a conventional IMformulation containing 2 μg HBsAg adsorbed to 50 μg AlPO₄(“liquid/commercial”). Titers are reported from weeks 4 and 6 sera.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified compositions or process parameters. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a particle” includes a mixture of two or more suchparticles, reference to “an excipient” includes mixtures of two or moresuch excipients, and the like.

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although a number of methodsand materials similar or equivalent to those described herein can beused in the practice of the present invention, the preferred materialsand methods are described herein.

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

As used herein, the term “pharmaceutical” or “pharmaceutical agent”intends any compound or composition of matter which, when administeredto an organism (human or animal) induces a desired pharmacologic and/orphysiologic effect by local and/or systemic action. The term thereforeencompasses those compounds or chemicals traditionally regarded asdrugs, as well as biopharmaceuticals including molecules such aspeptides, hormones, nucleic acids, gene constructs and the like. Moreparticularly, the term “pharmaceutical” or “pharmaceutical agent”includes compounds or compositions for use in all of the majortherapeutic areas including, but not limited to, anti-infectives such asantibiotics and antiviral agents; analgesics and analgesic combinations;local and general anaesthetics; anorexics; antiarthritics; antiasthmaticagents; anticonvulsants; antidepressants; antihistamines;anti-inflammatory agents; antinauseants; antineoplastics; antipruritics;antipsychotics; antipyretics; antispasmodics; cardiovascularpreparations (including calcium channel blockers, ACE-inhibitors,beta-blockers, beta-agonists and antiarrythmics); antihypertensives;diuretics; vasodilators; central nervous system stimulants; cough andcold preparations; decongestants; diagnostics; hormones; bone growthstimulants and bone resorption inhibitors; immunosuppressives; musclerelaxants; psychostimulants; sedatives; tranquilizers; therapeuticproteins (e.g., antigens, antibodies, growth factors, cytokines,interleukins, lymphokines, interferons, enzymes, etc.), peptides andfragments thereof (whether naturally occurring, chemically synthesizedor recombinantly produced); and nucleic acid molecules (polymeric formsof two or more nucleotides, either ribonucleotides (RNA) ordeoxyribonucleotides (DNA) including both double- and single-strandedmolecules, gene constructs, expression vectors, antisense molecules andthe like).

By “antigen” is meant a molecule which contains one or more epitopesthat will stimulate a host's immune system to make a cellularantigen-specific immune response or a humoral antibody response. Thus,antigens include polypeptides including antigenic protein fragments,oligosaccharides, polysaccharides and the like. Furthermore, the antigencan be derived from any known virus, bacterium, parasite, plant,protozoan or fungus, and can be a whole organism. The term also includestumor antigens. Similarly, an oligonucleotide or polynucleotide whichexpresses an antigen, such as in DNA immunization applications, is alsoincluded in the definition of an antigen. Synthetic antigens are alsoincluded, for example polyepitopes, flanking epitopes and otherrecombinant or synthetically derived antigens (Bergmann et al (1993)Eur. J. Immunol. 23:2777-2781; Bergmann et al. (1996) J. Immunol.157:3242-3249; Suhrbier, A. (1997) Immunol. and Cell Biol. 1:402-408;Gardner et al. (1998) 12^(th) World AIDS Conference, Geneva,Switzerland, Jun. 28-Jul. 3, 1998).

The above pharmaceuticals or pharmaceutical agents, alone or incombination with other agents, are typically prepared as pharmaceuticalcompositions which can contain one or more added materials such ascarriers, vehicles, and/or excipients. “Carriers,” “vehicles” and“excipients” generally refer to substantially inert materials which arenontoxic and do not interact with other components of the composition ina deleterious manner. These materials can be used to increase the amountof solids in particulate pharmaceutical compositions. Examples ofsuitable carriers include water, silicone, gelatin, waxes, and likematerials. Examples of normally employed “excipients,” includepharmaceutical grades of carbohydrates including monosaccharides,disaccharides, cyclodextrans, and polysaccharides (e.g., dextrose,sucrose, lactose, trehalose, raffinose, mannitol, sorbitol, inositol,dextrans, and maltodextrans); starch; cellulose; salts (e.g. sodium orcalcium phosphates, calcium sulfate, magnesium sulfate); citric acid;tartaric acid; glycine; high molecular weight polyethylene glycols(PEG); polyvinylpyrrolidone (PVP); Pluronics; surfactants; andcombinations thereof. Generally, when carriers and/or excipients areused; they are used in amounts ranging from about 0.1 to 99 wt % of thepharmaceutical composition.

The term “powder” as used herein refers to a composition that consistsof substantially solid particles that can be delivered transdermallyusing a needleless syringe device. The particles that make up the powdercan be characterized on the basis of a number of parameters including,but not limited to, average particle size, average particle density,particle morphology (e.g. particle aerodynamic shape and particlesurface characteristics) and particle penetration energy (P.E.).

The average particle size of the powders according to the presentinvention can vary widely and is generally from 0.1 to 250 μm, forexample from 10 to 100 μm and more typically from 20 to 70 μm. Theaverage particle size of the powder can be measured as a mass meanaerodynamic diameter (MMAD) using conventional techniques such asmicroscopic techniques (where particles are sized directly andindividually rather than grouped statistically), absorption of gases,permeability or time of flight. If desired, automatic particle-sizecounters can be used (e.g. Aerosizer Counter, Coulter Counter, HIACCounter, or Gelman Automatic Particle Counter) to ascertain the averageparticle size.

Actual particle density or “absolute density” can be readily ascertainedusing known quantification techniques such as helium pycnometry and thelike. Alternatively, envelope (“tap”) density measurements can be usedto assess the density of a powder according to the invention. Theenvelope density of a powder of the invention is generally from 0.5 to25 g/cm³, preferably from 0.8 to 1.5 g/cm³.

Envelope density information is particularly useful in characterizingthe density of objects of irregular size and shape. Envelope density isthe mass of an object divided by its volume, where the volume includesthat of its pores and small cavities but excludes interstitial space. Anumber of methods of determining envelope density are known in the art,including wax immersion, mercury displacement, water absorption andapparent specific gravity techniques. A number of suitable devices arealso available for determining envelope density, for example, theGeoPYc™ Model 1360, available from the Micromeritics Instrument Corp.The difference between the absolute density and envelope density of asample pharmaceutical composition provides information about thesample's percentage total porosity and specific pore volume.

Particle morphology, particularly the aerodynamic shape of a particle,can be readily assessed using standard light microscopy. It is preferredthat the particles which make up the instant powders have asubstantially spherical or at least substantially elliptical aerodynamicshape. It is also preferred that the particles have an axis ratio of 3or less to avoid the presence of rod- or needle-shaped particles. Thesesame microscopic techniques can also be used to assess the particlesurface characteristics, e.g. the amount and extent of surface voids ordegree of porosity.

Particle penetration energies can be ascertained using a number ofconventional techniques, for example a metallized film P.E. test. Ametallized film material (e.g. a 125 μm polyester film having a 350 Ålayer of aluminum deposited on a single side) is used as a substrateinto which the powder is fired from a needleless syringe (e.g. theneedleless syringe described in U.S. Pat. No. 5,630,796 to Bellhouse etal) at an initial velocity of about 100 to 3000 m/sec. The metallizedfilm is placed, with the metal-coated side facing upwards, on a suitablesurface.

A needleless syringe loaded with a powder is placed with its spacercontacting the film, and then fired. Residual powder is removed from themetallized film surface using a suitable solvent. Penetration energy isthen assessed using a BioRad Model GS-700 imaging densitometer to scanthe metallized film, and a personal computer with a SCSI interface andloaded with MultiAnalyst software (BioRad) and Matlab software (Release5.1, The Math Works, Inc.) is used to assess the densitometer reading. Aprogram is used to process the densitometer scans made using either thetransmittance or reflectance method of the densitometer. The penetrationenergy of the spray freeze-dried powders should be equivalent to, orbetter than that of reprocessed mannitol particles of the same size(mannitol particles that are freeze-dried, compressed, ground and sievedaccording to the methods of commonly owned International Publication No.WO 97/48485, incorporated herein by reference).

The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably herein and refer to a polymeric form of nucleotides ofany length, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three-dimensional structure, andmay perform any function, known or unknown. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term nucleic acid sequence is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

A “vector” is capable of transferring nucleic acid sequences to targetcells (e.g., viral vectors, non-viral vectors, particulate carriers, andliposomes). Typically, “vector construct”, “expression vector”, and“gene transfer vector”, mean any nucleic acid construct capable ofdirecting the expression of a gene of interest and which can transfergene sequences to target cells. Thus, the term includes cloning andexpression vehicles, as well as viral vectors. A “plasmid” is a vectorin the form of an extrachromosomal genetic element.

A nucleic acid sequence which “encodes” a selected antigen is a nucleicacid molecule which is transcribed (in the case of DNA) and translated(in the case of mRNA) into a polypeptide in vivo when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Forthe purposes of the invention, such nucleic acid sequences can include,but are not limited to, cDNA from viral, procaryotic or eucaryotic mRNA,genomic sequences from viral or procaryotic DNA or RNA, and evensynthetic DNA sequences. A transcription termination sequence may belocated 3′ to the coding sequence.

A “promoter” is a nucleotide sequence which initiates and regulatestranscription of a polypeptide-encoding polynucleotide. Promoters caninclude inducible promoters (where expression of a polynucleotidesequence operably linked to the promoter is induced by an analyte,cofactor, regulatory protein, etc.), repressible promoters (whereexpression of a polynucleotide sequence operably linked to the promoteris repressed by an analyte, cofactor, regulatory protein, etc.), andconstitutive promoters. It is intended that the term “promoter” or“control element” includes full-length promoter regions and functional(e.g., controls transcription or translation) segments of these regions.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a nucleic acidsequence is capable of effecting the expression of that sequence whenthe proper enzymes are present. The promoter need not be contiguous withthe sequence, so long as it functions to direct the expression thereof.Thus, for example, intervening untranslated yet transcribed sequencescan be present between the promoter sequence and the nucleic acidsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

The term “nucleic acid immunization” is used herein to refer to theintroduction of a nucleic acid molecule encoding one or more selectedantigens into a host cell for the in vivo expression of the antigen orantigens. The nucleic acid molecule can be introduced directly into therecipient subject by transdermal particle delivery. The moleculealternatively can be introduced ex vivo into cells which have beenremoved from a subject. In this latter case, cells containing thenucleic acid molecule of interest are re-introduced into the subjectsuch that an immune response can be mounted against the antigen encodedby the nucleic acid molecule. The nucleic acid molecules used in suchimmunization are generally referred to herein as “nucleic acidvaccines.”

The term “solids content” indicates the amount of solids which areeither dissolved or suspended in the solvent(s) used.

The term “subject” refers to any member of the subphylum cordataincluding, without limitation, humans and other primates includingnon-human primates such as chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The term does not denote a particularage. Thus, both adult and newborn individuals are intended to becovered. The methods described herein are intended for use in any of theabove vertebrate species, since the immune systems of all of thesevertebrates operate similarly.

The term “transdermal delivery” includes both transdermal(“percutaneous”) and transmucosal routes of administration, i.e.delivery by passage through the skin or mucosal tissue. See, e.g.,Transdermal Drug Delivery: Developmental Issues and ResearchInitiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989);Controlled Drug Delivery Fundamentals and Applications, Robinson and Lee(eds.), Marcel Dekker Inc., (1987); and Transdermal Delivery of Drugs,Vols. 1-3, Kydonieus and Berner (eds.), CRC Press, (1987).

B. General Methods

The invention is concerned with processes for producing powders suitablefor transdermal delivery via needleless syringe. As such, the particleswhich make up the powdered composition must have sufficient physicalstrength to withstand sudden acceleration to several times the speed ofsound and the impact with, and passage through, the skin and tissue.

In order to achieve powders having these properties, the process of thepresent invention is carried out using a solution or suspensioncontaining the desired pharmaceutical agent and having a total solidscontent of 20% by weight or more in the solvent system. Preferably thesolids content in the solvent system is 25% by weight or more, morepreferably 28% or 30% by weight or more and particularly preferably 40%by weight or more. The use of a solution or suspension having a solidscontent of 20% by weight or more minimises pore formation during thedrying process and increases the density of the particles formed. Thisincreased density is important in ensuring that the particles aresufficiently robust to survive the harsh conditions of transdermaldelivery via needleless syringe. The solids content of the solution ordispersion may be up to 50% by weight, up to 60% by weight or even up to70% by weight. The upper limit depends upon, for example, the particularcomponents of the solution or dispersion and the desired characteristicsof the resulting spray freeze-dried particles.

The pharmaceutical agent used to produce the powders of the inventionmay be any small molecule drug substance, organic or inorganic chemical,vaccine, or peptide (polypeptide and/or protein) of interest. Inparticular embodiments, the pharmaceutical agent is a biopharmaceuticalpreparation of a peptide, polypeptide, protein or any other suchbiological molecule. Exemplary peptide and protein formulations include,without limitation, insulin; calcitonin; octreotide; endorphin;liprecin; pituitary hormones (e.g., human growth hormone and recombinanthuman growth hormone (hGH and rhGH), HMG, desmopressin acetate, etc);follicle luteoids; growth factors (such as growth hormone releasingfactor (GHRF), somatostatin, somatotropin and platelet-derived growthfactor); asparaginase; chorionic gonadotropin; corticotropin (ACTH);erythropoietin (EPO); epoprostenol (platelet aggregation inhibitor);glucagon; interferons; interleukins; menotropins (urofollitropin, whichcontains follicle-stimulating hormone (FSH); and luteinizing hormone(LH)); oxytocin; streptokinase; tissue plasminogen activator (TPA);urokinase; vasopressin; desmopressin; ACTH analogues; angiotensin IIantagonists; antidiuretic hormone agonists; bradykinin antagonists; CD4molecules; antibody molecules and antibody fragments (e.g., Fab, Fab₂,Fv and sFv molecules); IGF-1; neurotrophic factors; colony stimulatingfactors; parathyroid hormone and agonists; parathyroid hormoneantagonists; prostaglandin antagonists; protein C; protein S; renininhibitors; thrombolytics; tumor necrosis factor (TNF); vaccines(particularly peptide vaccines including subunit and synthetic peptidepreparations); vasopressin antagonists analogues; and α-1 antitrypsin.Additionally, nucleic acid preparations, such as vectors or geneconstructs for use in subsequent gene delivery, can be used.

Particularly suitable pharmaceutical agents for use herein are antigens.Any suitable antigen as defined herein may be employed. The antigen maybe a viral antigen. The antigen may therefore be derived from members ofthe families Picornaviridae (e.g. polioviruses, etc.); Caliciviridae;Togaviridae (e.g. rubella virus, dengue virus, etc.); Flaviviridae;Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g. rabiesvirus, etc.); Filoviridae; Paramyxoviridae (e.g. mumps virus, measlesvirus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g.influenza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae;Retroviradae (e.g. HTLV-I; HTLV-II; HIV-1 and HIV-2); and simianimmunodeficiency virus (SIV) among others.

Alternatively, viral antigens may be derived from a papillomavirus (e.g.HPV); a herpesvirus; a hepatitis virus, e.g. hepatitis A virus (HAV),hepatitis B virus (HBV), hepatitis C (HCV), the delta hepatitis virus(HDV), hepatitis E virus (HEV) or hepatitis G virus (HGV); and thetick-borne encephalitis viruses. See, e.g. Virology, 3rd Edition (W. K.Joklik ed. 1988); Fundamental Virology, 2nd Edition (B. N. Fields and D.M. Knipe, eds. 1991) for a description of these viruses.

Bacterial antigens for use in the invention can be derived fromorganisms that cause diphtheria, cholera, tuberculosis, tetanus,pertussis, meningitis and other pathogenic states, includingMeningococcus A, B and C, Hemophilus influenza type B (HIB) andHelicobacter pylori. A combination of bacterial antigens may beprovided, for example diphtheria, pertussis and tetanus antigens.Suitable pertussis antigens are pertussis toxin and/or filamentoushemagglutinin and/or pertactin, alternatively termed P69. Ananti-parasitic antigen may be derived from organisms causing malaria andLyme disease.

Antigens for use in the present invention can be produced using avariety of methods known to those of skill in the art. In particular,the antigens can be isolated directly from native sources, usingstandard purification techniques. Alternatively, whole killed,attenuated or inactivated bacteria, viruses, parasites or other microbesmay be employed. Yet further, antigens can be produced recombinantlyusing known techniques. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Vols. I and II (D. N. Glover et.1985).

Antigens for use herein may also be synthesised, based on describedamino acid L sequences, via chemical polymer syntheses such as solidphase peptide synthesis. Such methods are known to those of skill in theart. See, e.g. J. M. Stewart and J. D. Young, Solid Phase PeptideSynthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984) and G.Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology,editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York,(1980), pp. 3-254, for solid phase peptide synthesis techniques; and M.Bodansky, Principles of Peptide Synthesis, Springer-Verlag Berlin (1984)and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis,Biology, supra, Vol. 1, for classical solution synthesis.

The pharmaceutical agent may alternatively be a nucleic acid molecule.The pharmaceutical agent can thus be a polynucleotide which expresses anantigen, such as in DNA immunization applications. An expression vectorcan thus be employed in which a nucleic acid sequence encoding a desiredpolypeptide such as an antigen is operably linked to a promoter.

Typically, the nucleic acid molecule comprises a therapeuticallyrelevant nucleotide sequence for delivery to a subjected. Thus, thenucleic acid molecule may comprise one or more genes encoding a proteindefective or missing from a target cell genome or one or more genes thatencode a non-native protein having a desired biological or therapeuticeffect (e.g., an antiviral function). The molecule may comprise asequence capable of providing immunity, for example an immunogenicsequence that serves to elicit a humoral and/or cellular response in asubject, or a sequence that corresponds to a molecule having anantisense or ribozyme function. For the treatment of genetic disorders,functional genes corresponding to genes known to be deficient in theparticular disorder can be administered to a subject.

Suitable nucleic acids for delivery include those used for the treatmentof inflammatory diseases, autoimmune, chronic and infectious diseases,including such disorders as AIDS, cancer, neurological diseases,cardivascular disease, hypercholestemia; various blood disordersincluding various anemias, thalassemia and hemophilia; genetic defectssuch as cystic fibrosis, Gaucher's Disease, adenosine deaminase (ADA)deficiency, emphysema, etc. A number of antisense oligonucleotides(e.g., short oligonucleotides complementary to sequences around thetranslational initiation site (AUG codon) of an mRNA) that are useful inanitsense therapy for cancer and for viral diseases have been describedin the art. See, e.g., Han et al (1991) Proc. Natl. Acad. Sci USA88:4313; Uhlmann et al (1990) Chem. Rev. 90:543, Helene et al (1990)Biochim. Biophys. Acta. 1049:99; Agarwal et al (1988) Proc. Natl. Acad.Sci. USA 85: 7079; and Heikkila et al (1987) Nature 328:445. A number ofribozymes suitable for use herein have also been described. See, e.g.,Chec et al (1992) J. Biol. Chem. 267:17479 and U.S. Pat. No. 5,225,347to Goldberg et al.

For example, in methods for the treatment of solid tumors, genesencoding toxic peptides (i.e., chemotherapeutic agents such as ricin,diptheria toxin and cobra venom factor), tumor suppressor genes such asp53, genes coding for mRNA sequences which are antisense to transformingoncogenes, antineoplastic peptides such as tumor necrosis factor (TNF)and other cytokines, or transdominant negative mutants of transformingoncogenes, can be delivered for expression at or near the tumor site.

Similarly, nucleic acids coding for peptides known to display antiviraland/or antibacterial activity, or stimulate the host's immune system,can also be administered. The nucleic acid may encode one of the variouscytokines (or functional fragments thereof), such as the interleukins,interferons, chemokines, chemotaxic factors, and colony stimulatingfactors. The nucleic acid may encode an antigen for the treatment orprevention of a number of conditions including but not limited tocancer, allergies, toxicity and infection by a pathogen such as, but notlimited to, fungus, viruses including Human Papiloma Viruses (HPV), HIV,HSV2/HSV1, influenza virus (types A, B and C), Polio virus, RSV virus,Rhinoviruses, Rotaviruses, Hepaptitis A virus, Norwalk Virus Group,Enteroviruses, Astroviruses, Measles virus, Par Influenza virus, Mumpsvirus, Varicella-Zoster virus, Cytomegalovirus, Epstein-Barr virus,Adenoviruses, Rubella virus, Human T-cell Lymphoma type I virus(HTLV-I), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis Dvirus, Pox virus, Marburg and Ebola; bacteria including M. tuberculosis,Chlamydia, N. gonorrheae, Shigella, Salmonella, Vibrio Cholera,Treponema pallidua, Pseudomonas, Bordetella pertussis, Brucella,Franciscella tulorensis, Helicobacter pylori, Leptospria interrogaus,Legionella pnumophila, Yersinia pestis, Streptococcus (types A and B),Pneumococcus, Meningococcus, Hemophilus influenza (type b), Toxoplamagondic, Complybacteriosis, Moraxella catarrhalis, Donovanosis, andActinomycosis; fungal pathogens including Candidiasis and Aspergillosis;parasitic pathogens including Taenia, Flukes, Roundworms, Amebas,Giardia species, Cryptosporidium, Schitosoma species, Pneumocystiscarinii, Trichuriasis species, and Trichinella species. The nucleic acidmy also be used to provide a suitable immune response against numerousveterinary diseases, such as Foot and Mouth diseases, Coronavirus,Pasteurella multocida, Helicobacter, Strongylus vulgaris, Actinobacilluspleuropneumonia, Bovine viral diarrhea virus (BVDV), Klebsiellapneumoniae, E. Coli, Bordetella pertussis, Bordetella parapertussis andbrochiseptica. Thus in one aspect, the particles of the presentinvention may find use as a vaccine.

The invention will also find use in antisense therapy, e.g., for thedelivery of oligonucleotides able to hybridize to specific complementarysequences thereby inhibiting the transcription and/or translation ofthese sequences. Thus DNA or RNA coding for proteins necessary for theprogress of a particular disease can be targeted, thereby disrupting thedisease process. Antisense therapy, and numerous oligonucleotides whichare capable of binding specifically and predictably to certain nucleicacid target sequences in order to inhibit or modulate the expression ofdisease-causing genes are known and readily available to the skilledpractitioner. Uhlmann et al (1990) Chem Rev. 90: 543, Neckers et al(1992) Int. Rev. Oncogenesis 3, 175; Simons et al (1992) Nature 159, 67;Bayever et al (1992) Antisense Res. Dev. 2: 109; Whitesell et al (1991)Antisense Res. Dev. 1: 343; Cook et al (1991) Anti-cancer Drug Design 6:585; Eguchi et al (1991) Ann. Rev. Biochem. 60: 631. Accordingly,antisense oligonucleotides capable of selectively binding to targetsequences in host cells are provided herein for use in antisensetherapeutics.

The antigen or other pharmaceutical agent employed in the presentinvention may optionally be adsorbed into an adjuvant, such as analuminum salt adjuvant or calcium salt adjuvant. Alternatively, theantigen or other pharmaceutical agent may be used without an adjuvant.Suitable adjuvants include aluminium hydroxide, aluminum phosphate,aluminum sulfate and calcium phosphate.

The pharmaceutical agent is typically prepared as an aqueouspharmaceutical composition using a suitable aqueous carrier, along withsuitable excipients, protectants, solvents, salts, surfactants,buffering agents and the like. Suitable excipients can include anymaterials that are innocuous when administered to an individual, and donot significantly interact with the pharmaceutical agent in a mannerthat alters its pharmaceutical activity. Examples of normally employedexcipients include, but are not limited to, pharmaceutical grades ofdextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol,dextran, starch, cellulose, sodium or calcium phosphates, calciumcarbonate, calcium sulfate, sodium citrate, citric acid, tartaric acid,glycine, high molecular weight polyethylene glycols (PEG), andcombinations thereof. Suitable solvents include, but are not limited to,methylene chloride, acetone, methanol, ethanol, isopropanol and water.Typically, water is used as the solvent. Generally pharmaceuticallyacceptable salts having molarities ranging from about 1 mM to 2M can beused. Pharmaceutically acceptable salts include, for example, mineralacid salts' such as hydrochlorides, hydrobromides, phosphates, sulfates,and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. A thorough discussionof pharmaceutically acceptable excipients, vehicles and auxiliarysubstances is available in REMINGTON'S PHARMACEUTICAL SCIENCES (MackPub. Co., N.J. 1991), incorporated herein by reference.

The excipients chosen for use in the present invention may servespecific functions such as protein stabilization or surface protectionor may be used as bulking agents or to maintain low hygroscopicity ofthe powders. Although the invention is not limited to the use of anyspecific excipients, particularly suitable excipients includesaccharides, which may be amorphous or crystalline saccharides, polymersor amino acids or physiologically acceptable salts thereof. The use ofthese particular excipient compositions allow the particles to collapseand densify during freezing and therefore provide powders which areparticularly suitable for injection via a needleless syringe. Thesaccharide may be a monosaccharide, disaccharide or higher oligo- orpoly-saccharide. The excipients may be selected from carbohydrates,sugars and sugar alcohols. Such excipients may be crystalline oramorphous.

Preferably, one, two or three of these additives are present in thesolution or suspension in amounts of at least 15% by weight, preferablyat least 20% by weight, such as at least 25%, 28% or 30% by weight andmore preferably at least 40% by weight. Such additives may be present inamounts of up to 50% by weight, up to 60% by weight or even up to 70% byweight. The upper limit depends upon, for example, the particularadditives used and the desired characteristics of the resulting sprayfreeze-dried particles. Most preferably one or two different excipientsare used.

Suitable amorphous saccharides include sugars. The amorphous excipientmay thus be selected from dextrose, sucrose, lactose, trehalose,cellobiose, raffinose, isomaltose and other carbohydrates such ascyclodextrins. Such sugars are capable of stabilizing proteins used aspharmaceutical agents during the spray-freeze-drying process and duringlong-term storage.

Suitable crystalline carbohydrates, sugars and sugar alcohols includemannitol, sorbitol and allitol. The combination of such a crystallineexcipient with an amorphous excipient, typically an amorphous sugar,encourages the collapse of particles during freeze-drying and aids theformation of dense particles.

Suitable polymers include polysaccharides such as dextran ormaltodextran; starch; cellulose; polyvinylpyrrolidone (PVP),particularly PVP K-17; polyvinyl alcohol (PVA); and polyethylene glycols(PEG), particularly PEG with a molecular weight of 8,000. Dextrans arepreferred. Various grades of dextran are available. A suitable dextranmay have a molecular weight of greater than about 15,000 such as fromabout 15,000 to about 45,000, from about 60,000 to about 90,000 or fromabout 100,000 to about 200,000. The addition of a polymer as anexcipient tends to provide powders with improved flowability and mayalso provide increased protein stability.

Suitable amino acids and physiologically acceptable salts of amino acidsinclude glycine, alanine, glutamine, arginine, lysine and histidine andsalts thereof such as alkali or alkaline earth metals salts such assodium, potassium or magnesium salts or salts with other amino acidssuch as glutamate or aspartate salts.

The most preferred combinations of excipients for use in the presentinvention include an amorphous saccharide with a crystalline saccharideand optionally also a polymer and/or an amino acid or a salt thereof.The excipients may comprise an amorphous saccharide which is typicallypresent in an amount of from 10 to 90% by weight, preferably from 50 to80% and more preferably from 60 to 75% by weight; and a crystallinesaccharide which is typically present in an amount of from 10 to 90% byweight, preferably from 20 to 50% and more preferably from 25 to 40% byweight. This combination of excipients is preferably used together witha surfactant which is typically present in an amount of from 1 to 5% byweight. Alternatively, the additives may comprise an amorphoussaccharide which is typically present in an amount of from 10 to 80% byweight, preferably from 20 to 50%, more preferably from 25 to 35% byweight; a crystalline saccharide which is typically present in an amountof from 10 to 80% by weight, preferably from 20 to 50%, more preferablyfrom 25 to 35% by weight; and a polymer or an amino acid or saltthereof, each of which is present in an amount of from 10 to 80% byweight, preferably from 30 to 60%, more preferably from 30 to 50% byweight.

The most preferred additive combinations include trehalose/mannitol,typically at a weight ratio of about 70/30; trehalose/mannitol/dextran,typically at a weight ratio of about 30/30/40; trehalose/mannitol/PVP ata weight ratio of about 30/30/40; trehalose/mannitol/PEG, typically at aweight ratio of about 30/30/40; or trehalose/mannitol/arginineglutamate, typically at a weight ratio of about 30/30/40. Particularlysuitable particles can be prepared from an aqueous solution ordispersion of a pharmaceutical agent which further comprises trehalose,mannitol and dextran in a weight ratio of from about 3:3.:4 to about4:4:3.

The use of these preferred additive combinations in the amountsdescribed above helps to provide particles with a high density. Thus,two major factors of the present invention act towards increasing thedensity of the particles. The first is the presence of at least 20% byweight of solids in the solution or suspension prior to freeze-dryingand the second is the selection of particular excipient compositions.

Whilst the excipient combinations described above are not essential foruse in the present invention, they are particularly preferred when thetotal amount of solids in the solution or suspension is close to 20% byweight, such as less than 40% by weight, in particular less than 30% orless than 25% by weight. When the solution or suspension has a solidscontent as low as this, the density of the particles, whilst sufficientfor the purposes of the invention, can desirably be increased further byuse of the above-described excipient compositions. However, if thesolids content is above 30% by weight or more preferably above 40% byweight, the particles produced will be sufficiently dense, so that theextra density obtained by using the preferred excipients in the ratiosdescribed above is less important.

The particles of the invention may additionally contain other additivessuch as surfactants. Suitable surfactants for use in the presentinvention include non-ionic surfactants. The surfactant may be apolysorbate such as Tween 20 and Tween-80, a Pluronics surfactant suchas F68 or a Span surfactant. The surfactant can be used in combinationwith any of the above-named combinations of excipients.

The particles of the invention are formed by first dissolving orsuspending the pharmaceutical agent, and any required additives, inwater. The aqueous solution or suspension formed must have a totalsolids content in the water of at least 20% by weight. The aqueoussolution or suspension is then spray freeze-dried. Any known techniquein the art (for example the methods described by Mumenthaler et al, Int.J. Pharmaceutics (1991) 72, pages 97-110 and Maa et al, Phar. Res.(1999) Vol. 16, page 249) may be used to carry out the sprayfreeze-drying step.

A typical spray freeze-drying technique involves atomising the aqueoussolution or suspension into a liquified gas, which is generally understirring. The liquified gas can be liquid argon, liquid nitrogen, or anyother gas that results in the immediate freezing of the atomiseddroplets of the aqueous solution or suspension. Preferably the liquifiedgas is an inert liquified gas such as liquid nitrogen.

The liquified gas containing the frozen droplets of the aqueous solutionor suspension is then freeze-dried. It is not contacted with an organicsolvent such as methanol, ethanol, ethyl ether, acetone, pentane,methylene chloride, chloroform or ethyl acetate. Drying is not thereforeconducted according to the procedures described in U.S. Pat. No.5,019,400.

Typically, the liquified gas containing the frozen droplets istransferred into a lyophiliser for freeze-drying. The liquified gascontaining the frozen droplets is usually poured into a metal tray andintroduced into the lyophiliser. The frozen droplets are freeze-dried inthe tray. The liquid nitrogen evaporates and the frozen water containedin the droplets is removed by sublimation. The resulting particles arecollected. They can be washed as desired.

In more detail, the liquified gas containing frozen droplets of theatomized solution or suspension is held at reduced temperature, forexample from about −60° C. to −40° C. Typically, that is followed bytwo-stage vacuum drying preferably under a pressure of from about 20 to500 mT (2.666 to 66.65 Pa). The first drying stage is normally performedat a reduced temperature such as from about −50° C. to 0° C., for aperiod of about 4 to 24 hours. Frozen water is removed by icesublimation. In the second drying stage, drying is normally performed ata higher temperature such as from about 5 to 30° C. at a lower pressure,preferably less than 100 mT down to about 10 mT, for a period of about 5to 24 hours. The precise spray freeze-drying conditions used may beselected according to the desired properties of the particles to beproduced. Thus, the temperatures, pressures and other conditions may bevaried as desired.

Preferably, the nozzle used to atomise the solution or suspension is anultrasonic nozzle. This has the advantage of being a mild process whichgenerates little stress to the biomolecules which are frequently used astherapeutic agents in the present invention. In addition, use of anultrasonic nozzle eliminates the need for pressurized gas to assist theliquid feed which, in turn helps increase the yield of the process. Thepredominant variable for control of droplet size in an ultrasonic nozzlesystem is the nozzle frequency, although surface tension, viscosity anddensity of the liquid feed are additional variables that can bemanipulated to control droplet size. Thus, for example, smallerparticles may be produced by increasing the nozzle frequency and viceversa. When using the ultrasonic nozzle system. am accurate. low-pulsefeed pump can be used to delivery the liquid feed, wherein such pumpsare particularly well suited when operating at low feed rates (e.g.,about 3 to 5 ml per minute) normally associated with laboratory-scaleparticle production. It has been found that atomization proceeds well atabout 1 to 2 Watts above the “critical power” level of the low-pulsepump system. In some drying cycles, it has been found that operation atabout 2.9 to 3.1 Watts allows for the most efficient atomization,however the exact operating conditions will also depend upon the liquidcharacteristics of the feed (viscosity, density, total solids content,surface tension, etc.).

A dual spray freeze-drying process may also be used. This process isparticularly useful when the pharmaceutical agent is a protein having alow water solubility. This dual process comprises spray freeze-dryingthe liquid protein to form a dry powder. This powder is thenreconstituted in water to provide a suspension having the desired solidcontent and spray freeze-dried for a second time.

The spray freeze-dried particular that are obtained according to theinvention can be collected, washed and dried. The dried particles canthen be sieved to obtained particles of the desired size.

The particles of the invention have a size appropriate for high-velocitytransdermal delivery to a subject, typically across the stratum corneumor a transmucosal membrane. The mass mean aerodynamic diameter (MMAD) ofthe particles is from about 0.1 to 250 μm. The MMAD may be from 5 to 100μm or from 10 to 100 μm, preferably from 10 to 70 μm or from 20 to 70μm. Generally, less than 10% by weight of the particles have a diameterwhich is at least 5 μm more than the MMAD or at least 5 μm less than theMMAD. Preferably, no more than 5% by weight of the particles have adiameter which is greater than the MMAD by 5 μm or more.

Also preferably, no more than 5% by weight of the particles have adiameter which is smaller than the MMAD by 5 μm or more. The particlesize is controllable by varying the frequency of the ultrasonic nozzleused to atomise the solution or suspension. The particles typically havean envelope density of from 0.5 to 25 g/cm³, preferably from 0.6 to 1.8g/cm³. More preferably the envelope density is from 0.7 to 1.5 g/cm³.The attainment of the above minimum envelope density value isparticularly preferred, since particles with a lower density tend toperform poorly during penetration of the skin and may not be suitablefor use in a transdermal needleless injection system. The particles havea low porosity, wherein typically at least 70%, at least 80%, at least85% or at least 90% of the particle is nor occupied by pores.

While the shape of the individual particles may vary when viewed under amicroscope, the particles are preferably substantially spherical. Theaverage ratio of the major axis:minor axis is typically from 3:1 to 1:1,for example from 2:1 to 1:1.

The individual particles of the powder have a substantially sphericalaerodynamic shape with a substantially uniform, nonporous surface. Theparticles will also have a particle penetration energy suitable fortransdermal delivery from a needleless syringe device. The particlesshould also be free-flowing under a dry environment. For example, theparticles should flow freely in a vial upon rotation at a relativehumidity of less than 30%. Preferably, the particles are free-flowingunder ambient conditions, such as a relative humidity of less than 60%.The moisture content of the particles should preferably be less than 5%,more preferably less than 2%, after freeze-drying, and this level ofmoisture should be maintained during storage at less than 30% humidityfor, for example, at least one month and preferably much longer.

A detailed description of needleless syringe devices useful in thisinvention is found in the prior art, as discussed herein. These devicesare referred to as needleless syringe devices and representative ofthese devices are the dermal PowderJect® needleless syringe device andthe oral PowderJect® needleless syringe device (PowderJect TechnologiesLimited, Oxford, UK). By using these devices, an effective amount of thepowder of the invention is delivered to the subject. An effective amountis that amount needed to deliver a sufficient quantity of the desiredantigen to achieve vaccination. This amount will vary with the nature ofthe antigen and can be readily determined through clinical testing basedon known activities of the antigen being delivered. The “Physicians DeskReference” and “Goodman and Gilman's The Pharmacological Basis ofTherapeutics” are useful for the purpose of determining the amountneeded.

Needleless syringe devices for delivering particles were first describedin commonly owned U.S. Pat. No. 5,630,796 to Bellhouse et al,incorporated herein by reference. Although a number of specific deviceconfigurations are now available, such devices are typically provided asa pen-shaped instrument containing, in linear order moving from top tobottom, a gas cylinder, a particle cassette or package, and a supersonicnozzle with an associated silencer element. An appropriate powder (inthe present case, a spray freeze-dried powder of the invention) isprovided within a suitable container, e.g., a cassette formed by tworupturable-polymer membranes that are heat-sealed to a washer-shapedspacer to form a self-contained sealed unit. Membrane materials can beselected to achieve a specific mode of opening and burst pressure thatdictate the conditions at which the supersonic flow is initiated. Inoperation, the device is actuated to release the compressed gas from thecylinder into an expansion chamber within the device. The released gascontacts the particle cassette and, when sufficient pressure is builtup, suddenly breaches the cassette membranes sweeping the particles intothe supersonic nozzle for subsequent delivery. The nozzle is designed toachieve a specific gas velocity and flow pattern to deliver a quantityof particles to a target surface of predefined area. The silencer isused to attenuate the noise produced by the transient supersonic flowand/or membrane rupture.

A second needleless syringe device for delivering particles is describedin commonly owned International Publication No. WO 96/20022. Thisdelivery system also uses the energy of a compressed gas source toaccelerate and deliver powdered compositions; however, it isdistinguished from the system of U.S. Pat. No. 5,630,796 in its use of ashock wave instead of gas flow to accelerate the particles. Moreparticularly, an instantaneous pressure rise provided by a shock wavegenerated behind a flexible dome strikes the back of the dome, causing asudden eversion of the flexible dome in the direction of a targetsurface. This sudden eversion catapults a powdered composition (which islocated on the outside of the dome) at a sufficient velocity, thusmomentum, to penetrate target tissue, e.g., oral mucosal tissue. Thepowdered composition is released at the point of full dome eversion. Thedome also serves to completely contain the high-pressure gas flow, whichtherefore does not come into contact with the tissue. Because the gas isnot released during this delivery operation, the system is inherentlyquiet. This design can be used in other enclosed or otherwise sensitiveapplications for example, to deliver particles to sites reached byminimally invasive surgery.

In yet a further aspect of the invention, single unit dosages ormultidose containers, in which the powder of the invention may bepackaged prior to use, can comprise a hermetically sealed containerenclosing a suitable amount of the powder that makes up a suitable dose.The powder can be packaged as a sterile formulation, and thehermetically sealed container can thus be designed to preserve sterilityof the formulation until use. If desired, the containers can be adaptedfor direct use in the above-referenced needleless syringe systems.

Powders of the present invention can thus be packaged in individual unitdosages for delivery via a needleless syringe. As used herein, a “unitdosage” intends a dosage receptacle containing a therapeuticallyeffective amount of a powder of the invention. The dosage receptacletypically fits within a needleless syringe device to allow fortransdermal delivery from the device. Such receptacles can be capsules,foil pouches, sachets, cassettes or the like.

The container in which the powder is packaged can further be labeled toidentify the composition and provide relevant dosage information. Inaddition, the container can be labeled with a notice in the formprescribed by a governmental agency, for example the U.S. Food and DrugAdministration, wherein the notice indicates approval by the agencyunder U.S. Federal Law of the manufacture, use or sale of the powdercontained therein for human administration.

The actual distance which the delivered particles will penetrate atarget surface depends upon particle size (e.g., the nominal particlediameter assuming a roughly spherical particle geometry), particledensity, the initial velocity at which the particle impacts the surface,and the density and kinematic viscosity of the targeted skin tissue. Inthis regard, optimal particle densities for use in needleless injectiongenerally range between about 0.5 and 25 g/cm³, preferably between about0.7 and 1.5 g/cm³, and injection velocities generally range betweenabout 100 and 3,000 m/sec. With appropriate gas pressure, particleshaving an average diameter of 10-70 μm can be accelerated through thenozzle at velocities approaching the supersonic speeds of a driving gasflow.

If desired, the needleless syringe systems can be provided in apreloaded condition containing a suitable dosage of the powder of theinvention. The loaded syringe can be packaged in a hermetically sealedcontainer, which may further be labeled as described above.

A number of novel test methods have been developed, or established testmethods modified, in order to characterize performance of a needlelesssyringe device. These tests range from characterization of the powderedcomposition, assessment of the gas flow and particle acceleration,impact on artificial or biological targets, and measures of completesystem performance. One, several or all of the following tests can thusbe employed to assess the physical and functional suitability of thepowder of the invention for use in a needleless syringe system.

Assessment of Effect on Artificial Film Targets

A functional test that measures many aspects of powder injection systemssimultaneously has been designated as the “metallized film” or“penetration energy” (PE) test. It is based upon the quantitativeassessment of the damage that particles can do to a precision thin metallayer supported by a plastic film substrate. Damage correlates to thekinetic energy and certain other characteristics of the particles. Thehigher the response from the test (i.e., the higher the filmdamage/disruption) the more energy the device has imparted to theparticles. Either electrical resistance change measurement or imagingdensitometry, in reflectance or transmission mode, provide a reliablemethod to assess device or formulation performance in a controllable andreproducible test.

The film test bed has been shown to be sensitive to particle deliveryvariations of all major device parameters including pressure, dose,particle size distribution and material, etc. and to be insensitive tothe gas. Aluminum of about 350 Angstrom thickness on a 125 μm polyestersupport is currently used to test devices operated at up to 60 barhelium pressure.

Assessment of Impact Effect on Engineering Foam Targets

Another means of assessing particle performance when delivered via aneedleless syringe device is to gauge the effect of impact on a rigidpolymethylimide foam (Rohacell 5 IIG, density 52 kg/m³, Rohm Tech Inc.,Malden, Mass.). The experimental set-up for this test is similar to thatused in the metallized film test. The depth of penetration is measuredusing precision calipers. For each experiment a processed mannitolstandard is run as comparison and all other parameters such as devicepressure, particle size range, etc., are held constant. Data also showthis method to be sensitive to differences in particle size andpressure. Processed mannitol standard as an excipient for drugs has beenproven to deliver systemic concentrations in preclinical experiments, sothe relative performance measure in the foam penetration test has apractical in vivo foundation. Promising powders can be expected to showequivalent or better penetration to mannitol for anticipation ofadequate performance in preclinical or clinical studies. This simple,rapid test has value as a relative method of evaluation of powders andis not intended to be considered in isolation.

Particle Attrition Test

A further indicator of particle performance is to test the ability ofvarious candidate compositions to withstand the forces associated withhigh-velocity particle injection techniques, that is, the forces fromcontacting particles at rest with a sudden, high velocity gas flow, theforces resulting from particle-to-particle impact as the powder travelsthrough the needleless syringe, and the forces resulting fromparticle-to-device collisions also as the powder travels through thedevice. Accordingly, a simple particle attrition test has been devisedwhich measures the change in particle size distribution between theinitial composition, and the composition after having been deliveredfrom a needleless syringe device.

The test is conducted by loading a particle composition into aneedleless syringe as described above, and then discharging the deviceinto a flask containing a carrier fluid in which the particularcomposition is not soluble (e.g., mineral oil, silicone oil, etc.). Thecarrier fluid is then collected, and particle size distribution in boththe initial composition and the discharged composition is calculatedusing a suitable particle sizing apparatus, e.g., an AccuSizer® model780 Optical Particle Sizer. Compositions that demonstrate less thanabout 50%, more preferably less than about 20% reduction in mass meandiameter (as determined by the AccuSizer apparatus) after deviceactuation are deemed suitable for use in the needleless syringe systemsdescribed herein.

Delivery to Human Skin In Vitro and Transepidermal Water Loss

For a powder performance test that more closely parallels eventualpractical use, candidate powder compositions can be injected intodermatomed, full thickness human abdomen skin samples. Replicate skinsamples after injection can be placed on modified Franz diffusion cellscontaining 32° C. water, physiologic saline or buffer. Additives such assurfactants may be used to prevent binding to diffusion cell components.Two kinds of measurements can be made to assess performance of theformulation in the skin.

To measure physical effects, i.e. the effect of particle injection onthe barrier function of skin, the transepidermal water loss (TEWL) canbe measured. Measurement is performed at equilibrium (about 1 hour)using a Tewameter TM 210® (Courage & Khazaka, Koln, Germany) placed onthe top of the diffusion cell cap that acts like a ˜12 mm chimney.Larger particles and higher injection pressures generate proportionallyhigher TEWL values in vitro and this has been shown to correlate withresults in vivo. Upon particle injection in vitro TEWL values increasedfrom about 7 to about 27 (g/m²h) depending on particle size and heliumgas pressure. Helium injection without powder has no effect. In vivo,the skin barrier properties return rapidly to normal as indicated by theTEWL returning to pretreatment values in about 1 hour for most powdersizes. For the largest particles, 53-75 μm, skin samples show 50%recovery in an hour and full recovery by 24 hours.

Delivery to Human Skin In Vitro and Drug Diffusion Rate

To measure the formulation performance in vitro, the drug or antigencomponent(s) of candidate powders can be collected by complete oraliquot replacement of the Franz cell receiver solution at predeterminedtime intervals for chemical assay using HPLC or other suitableanalytical technique. Concentration data can be used to generate adelivery profile and calculate a steady state permeation rate. Thistechnique can be used to screen formulations for early indication ofdrug or antigen binding to skin, drug or antigen dissolution, efficiencyof particle penetration of stratum corneum, etc., prior to in vivostudies.

These and other qualitative and quantitative tests can be used to assessthe physical and functional suitability of the present powders for usein a high-velocity particle injection device. It is preferred, thoughnot required, that the particles of a powder have the followingcharacteristics: a substantially spherical shape (e.g. an aspect ratioas close as possible to 1); a smooth surface; a suitable active loadingcontent; less than 20% reduction in particle size using the particleattrition test; an envelope density as close as possible to the truedensity of the constituents (e.g. greater than about 0.5 g/ml); and aMMAD of about 20 to 70 μm with a narrow particle size distribution. Thecompositions are typically free-flowing (e.g. free-flowing after 8 hoursstorage at 50% relative humidity and after 24 hours storage at 40%relative humidity). All of these criteria can be assessed using theabove-described methods, and are further detailed in the followingpublications, incorporated herein by reference. Etzler et al (1995)Part. Part. Syst. Charact. 12:217; Ghadiri, et al (1992) IFPRI FinalReport, FRR 16-03 University of Surrey, UK; Bellhouse et al (1997)“Needleless delivery of drugs in dry powder form, using shock waves andsupersonic gas flow,” Plenary Lecture 6, 21^(st) International Symposiumon Shock Waves, Australia; Kwon et al (1998) Pharm. Sci. suppl.1 (1),103; and Burkoth et al. (1999) “Transdermal and Transmucosal PowderedDrug Delivery,” in Critical Reviews in the Therapeutic Drug CarrierSystems 16(4):331-384, STephen Bruck Ed., Begell House Inc., New York,N.Y.

A powder of the invention may alternatively be used to vaccinate asubject via other routes. For this purpose, the powder may be combinedwith a suitable carrier or diluent such as Water for Injections orphysiologically saline. The resulting vaccine composition is typicallyadministered by injection, for example subcutaneously orintramuscularly.

Whichever route of administration is selected, an effective amount ofantigen is delivered to the subject being vaccinated. Generally from 50ng to 1 mg and more preferably from 1 μg to about 50 μg of antigen willbe useful in generating an immune response. The exact amount necessarywill vary depending on the age and general condition of the subject tobe treated, the particular antigen or antigens selected, the site ofadministration and other factors. An appropriate effective amount can bereadily determined by one of skill in the art.

Dosage treatment may be a single dose schedule or a multiple doseschedule. A multiple dose schedule is one in which a primary course ofvaccination may be with 1-10 separate doses, followed by other dosesgiven at subsequent time intervals, chosen to maintain and/or reinforcethe immune response, for example at 1-4 months for second dose and, ifneeded, a subsequent dose(s) after several months. The dosage regimenwill also, at least in part, be determined by the need of the subjectand be dependent on the judgement of the practitioner. Vaccination willof course generally be effected prior to primary infection with thepathogen against which protection is desired.

C. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Spray Freeze Drying (“SFD”) of Hepatitis B VaccineCompositions

1.1 Objectives:

-   -   To assess the SFD method for preparing Hepatitis-B vaccine        powders and to particularly assess the following formulation        parameters: (a) the concentration effect on vaccine; and (b) the        solid content effect on particle density.        1.2 Materials:

Hepatitis B surface antigen (HBsAg, Lot # 64850) was obtained from RheinAmericana S.A. (Argentine Republic). Nominal concentration of theantigen was reported as 1.37 mg/mL in the attached Quality ControlCertificate of Analysis. The materials used in the study are reportedbelow in Table 1.1. TABLE 1.1 Materials used in the study Material Lot #Source Comment Hepatitis-B 64850 Rhein Americana S.A. Nominal surface(Argentina Republic) concentration at antigen 1.37 mg/mL (20 μg perhuman dose), (“HBsAg”) Alum-adjuvanted 17 Rhein Americana S.A. 20 μgHBsAg HBsAg (Argentina Republic) adsorbed to 0.44 mg of alum per vial(˜1.0 mL), (“HBsAg/alum”) Dextran 69H1273 Sigma (St. Louis, MO) (MW 10kDa) Mannitol 127H0960 Sigma (St. Louis, MO) trehalose 28H3797 Sigma(St. Louis, MO) dihydrate1.3 Formulations:

Table 1.2, below, shows the percent (w/w) composition of the startingliquid formulations, and the targeted composition in 1.0-mg of payload.The key excipients were trehalose, mannitol, and dextran of 10 kDamolecular weight. Trehalose was used since it is capable of stabilizingthe protein during lyophilization and long-term storage, and thecombination of mannitol and trehalose allows dense particles to beproduced during spray-freeze-drying. Dextran was used to improve thepowder's flowability, the mechanical properties of the particles, andpossibly the overall vaccine composition stability. All liquidformulations were targeted to have a total solid content of 30%. TABLE1.2 Composition (% w/w) of HBsAg and HBsAg/alum formulations Batch #138-20-1 138-20-2 138-20-3 138-20-4A 138-20-4B 138-16-1 138-20-5 Solidcontent (%) 30 30 30 30 5 30 10 Conc. of HBsAg 403.5 407.5 403.5 403.5403.5 15.0 5.0 (human dose/mL) Batch size (dose) 500 500 500 400 300 400300 HBsAg solution 133.2 317.0 622.4 989.7 742.3 1891.0 5400.0 (mg)Trehalose 164.5 164.4 163.4 143.1 107.3 251.2 188.0 (dihydrate) Mannitol199.8 199.23 198.0 172.7 129.5 303.9 228.0 Dextran (10 kDa) 150.0 148.7148.5 129.1 96.9 228.9 172.21.4 Methods:

1.4.1 Vaccine Concentration

A centrifugal filter device, Centriprep, with a 10 kD limit regeneratedcellulose membrane and a 15 mL sample container (Millipore, Bedford,Mass.) was used to concentrate the alum-free bulk vaccine. The filterwas rinsed twice with 15 mL of nanopure water to remove the trace amountof glycerin present in the filter by centrifugation at 3000 rpm at 5-10°C. (Allegra 6®, Beckman, Fullerton, Calif.). The filter was then rinsedonce with a blank buffer at the specified pH. The vaccine solution wascentrifuged until a desired amount of permeate was collected. Threeseparate batches were prepared for three powder formulations.

The alum-adjuvanted bulk vaccine was pooled from vials and centrifuged(Allegra 6R, Beckman, Palo Alto, Calif.) at 3,000 rpm for 10 minutes.The cake was re-suspended with fresh water by vortex prior toformulating with excipients.

1.4.2 Spray-Freeze-Drying

The SFD apparatus featured an ultrasonic atomizer (Sono-Tek Corporation,Milton, N.Y.) having a spraying nozzle (Model #05793) and a power supply(Model #06-05108). The nozzle was equipped with a quasi-electric quartzcrystal capable of vibrating at a specific frequency that determines thesize of the droplets. The frequency of 60 kHz produces droplets mostlywithin the range of 20-80 μm. Circular metal pans (16-cm in diameter by6-cm in height) were used to contain the liquid nitrogen. For thelyophilization, a shelf freeze dryer (Model #TDS2C2B5200, Dura-Stop, FTSSystem, Stone Ridge, N.Y.) was used. This dryer can hold six metal pansin one batch run. Other apparatus included a magnetic stirrer withmagnetic stir bars, and a peristaltic pump (Model #77120-70, MasterFlexC/L, Barnant Company, Barrington, Ill.).

The liquid feed (vaccine formulation) was delivered by the peristalticpump at a flow rate of 1.5 mL/min into the ultrasonic atomizer (60 kHz)where the liquid formulation was sprayed into the liquid N₂-containingpan. After spraying, the pan containing frozen particles in liquidnitrogen was transferred to a pre-cooled freeze dryer (−55° C.). Liquidnitrogen evaporated in a few minutes. The freeze-drying cycle was variedto control particle characteristics, but a typical lyophilization cycleis listed below in Table 1.3. TABLE 1.3 Freeze-drying cycle Stage/CycleConditions Freezing pre-cool shelf temperature (ST) = −50° C. ramp at1.0° C./min to ST = −55° C., hold for 15 min wait for product temp (PT)= −48° C., hold for 120 min Primary Drying condenser/vacuum (C/V)switched “on” when condenser temp. reaches −40 C, vacuum pump turned onwait for chamber vacuum to reach 150 mT wait for foreline vacuum toreach 100 mT ramp at 1.0° C./min to ST = −10° C., hold for 24 hoursSecondary ramp at 1.0° C./min to ST = 20° C., hold for 24 hours Drying

After drying, the powder-containing pan was transferred into a dry boxpurged with nitrogen with relative humidity initially held at <20%. Therelative humidity was increased gradually to ˜40% to equilibrate thepowder prior to powder collection.

1.5 Powder Characterization:

1.5.1 Particle Size Analysis

The mean geometric diameter of the particles in the volume distributionwas determined using an AccuSizer 780 (Particle Sizing Systems, SantaBarbara, Calif.). Based on the light obscuration technique, theAccuSizer determines the particle size distribution without assuming theshape distribution of the particle. In addition, the size of theparticle population between 10% and 90% (volume) was also determined.Each analysis required approximately 5 mg of the powder sample. Powdersamples were suspended in light mineral oil and sonicated for 5-10seconds to remove agglomeration of particles before analysis.

The mean aerodynamic diameter of the particles in the volumedistribution was also determined using a dry powder dispersion-basedparticle size analyser (Aerosizer, API). Here again, each analysisrequired approximately 5 mg of the powder sample.

As reported below in Table 1.4, the particle size of all powders testedfell in the range of 20-60 μm based on Aerosizer measurement. TABLE 1.4Particle size result by Aerosizer D50% (median) D10% D90% Batch #Formulation size (μm) (μm) (μm) 138-20-1  2 μg HBsAg 40.1 25.1 57.4138-20-2  5 μg HBsAg 39.4 25.3 53.1 138-20-3 10 μg HBsAg 36.7 23.6 51.7138-20-4A 20 μg HBsAg 36.9 24.2 50.5 138-20-4B 20 μg HBsAg (FD/C/S) 39.826.9 54.6 138-16-1  2 μg HBsAg/50 μg alum 41.0 26.2 55.9 138-20-5C  2 μgHBsAg/50 μg g 42.9 31.3 54.3 alum SFD/C/S 138-16-1C  2 μg HBsAg/50 μgalum na na na SFD/sieved

1.5.2 Image Analysis

Visual analysis of the particles was performed using an opticalmicroscope (Model DMR, Leica, Germany) with 10×-eyepiece lens and10×-objective lens. The system was equipped with a Polaroid camerasystem for image output. Digital images were captured and stored.

The image pictures of selected formulations are presented in FIGS. 1 a-1d. As can be seen, particle shape was generally spherical, and theestimated particle size consistent with that measured by the Aerosizer.

1.5.3 Scanning Electron Microscopy (SEM)

SEM was performed on an Amray 1810T instrument after powder samples weresputter-coated with gold. Measurement was courtesy of Prof. Geoffrey Leeand Ms. Christine Sonner of the Friedrich-Alexander University inErlangen, Germany.

The SEM micrographs are presented in FIGS. 2 a-2 d. As can be seen, allSFD particles (FIGS. 2 a and 2 b) were spherical in shape with wrinkledmorphology, suggesting particle shrinkage during drying, while theparticles that were formed by a compress, grind and sieve (C/G/S)technique had an irregular shape and surface morphology.

1.5.4 Tap Density

Each powder sample was weighed in a glass vial and gently tapped againstthe lab bench for 20 times. By visual inspection, water of an equivalentvolume to that of the powder was placed into an empty vial of the sametype. The tap density of the powder sample could be calculated bydividing the powder sample weight with the water sample weight (assumingwater density=1 g/mL).

As summarized in Table 1.5 below, the tap density of all powderswas >0.4 g/mL. Higher tap densities are achievable by using a highersolid content, for example, tap densities of >0.6 g/mL were seen whenthe solid content was raised to 35%. TABLE 1.5 Tap density result Tapdensity Batch # Formulation (g/mL) 138-20-1  2 μg HBsAg 0.52 138-20-2  5μg HBsAg 0.51 138-20-3 10 μg HBsAg 0.43 138-20-4A 20 μg HBsAg 0.44138-20-4B 20 μg HBsAg (FD 0.49 and C/G/S) 138-16-1  2 μg HBsAg/50 μg0.51 alum 138-20-5C  2 μg HBsAg/50 μg 0.42 alum SFD and C/G/S 138-16-1C 2 μg HBsAg/50 μg 0.51 alum SFD/sieved

1.5.5 Moisture Content Analysis

Approximately 4-5 mg of the powder sample was recovered from thetrilaminate cassettes and transferred into an aluminium weighing-vesseland the weight was recorded. The sample was loaded into a Karl FisherCoulometer (Model 737, Brinkmann) equipped with a drying oven (Model707). The sample was heated to 150° C. for 150 sec with a gas flow rateof 100 mL/min within the drying oven.

As reported below in Table 1.6, the moisture content in the powdersranged from 3.3% to 4.1%. Under the same drying condition, there was noclear correlation between moisture content and chemical composition.TABLE 1.6 Moisture content result Moisture content Batch # Formulation(%) 138-20-1  2 μg HBsAg 3.9 138-20-2  5 μg HBsAg 3.6 138-20-3 10 μgHBsAg 4.1 138-20-4A 20 μg HBsAg 3.7 138-20-4B 20 μg HBsAg (FD/C/S) 3.3138-16-1  2 μg HBsAg/50 μg alum 3.9 138-20-5C  2 μg HBsAg/50 μg alum 3.3SFD/C/S 138-16-1C  2 μg HBsAg/50 μg alum 3.9 SFD/sieved

1.5.6 Particle Attrition Testing

This method allows particle attrition arising from, e.g., particlecollisions within the powder injection device to be quantified. Thismethod can be used to assess particle integrity upon contacting the skinprior to penetration indirectly through measuring the mean particle sizereduction and particle size distribution changes of the powder afterfiring from a needleless syringe (powder injection) device. The controlsample (prior to firing) was prepared by suspending 5 mg of powder inmineral oil (about 30 mL) in a 40 mL container. The mixture wasvortexed/sonicated to make a homogeneous suspension. The particle sizedistribution was measured using a particle size analyzer (AccuSizer 780,Particle Sizing Systems, Santa Barbara, Calif.). For the post-firingsample, the powder was actuated from the device (PowderJect® powderinjection device, PowderJect Pharmaceuticals plc, Oxford, UnitedKingdom), using trilaminate cassettes and 20 μm polycarbonate membranesto contain the powder, 5 shots at a payload of 2 mg each) into a 1 LErlenmeyer flask. The flask was coated with 25 mL of light mineral oiland the top was covered with a latex sheet with a 3/16 inch hole in thecenter. The flask sat for about 2-3 minutes until no flying particlescould be seen. The interior wall of the flask was washed with 25 mL offresh mineral oil and vortexed or shaken vigorously to establish ahomogeneous suspension. Fifteen mL of the powder suspension was furtherdiluted with 15-mL of mineral oil and dispensed into a 40 cc tube. Bothsamples before and after attrition were subject to light obscurationanalysis. The experiment was repeated three times and each sample wasmeasured in triplicate. The particle size distribution profile from thepost-firing sample was compared with that from the control sample. Thedecrease in the ratio of the respective mean size represents the extentof particle attrition.

As reported below in Table 1.7, particle size reduction for 3 selectedformulations was found to be similar and all less than 30%. TABLE 1.7Particle attrition test results Mean Particle Size of pre- Mean ParticleSize of % Reduction Samples Actuation(μm) post-Actuation(μm) in MeanSize 138-20-3 53.2 41.8 21.5 138-20-4A 53.9 41.4 23.2 1380-20-4B 55.340.2 27.3

1.5.7 Reconstitution of Alum-Containing Powder

The alum-containing powder sample (2-mg) was dissolved (reconstituted)in 0.5-mL of water. The optical image of the liquid suspension wastaken. This procedure allows alum coagulation to be determined.

Two alum-containing formulations (batch numbers 138-16-1 & 138-20-5C)were reconstituted in water and their optical micrographs are shown inFIGS. 3 a and 3 b. The sandy appearance of batch no. 138-16-1 (FIG. 3 a)suggests that alum coagulation after SFD is minimal. This is due thefast—freezing phenomenon associated with the SFD process. There isslight coagulation seen with batch no. 138-20-5C (FIG. 3 b) that has thesame formulation as batch 138-16-1 but has been further processed usinga compress, grind, and sieve technique. This is consistent with aprevious observation that compression caused alum to coagulate slightly.

1.5.8 SDS-PAGE Analysis

Coomassie colloidal stained SDS-polyacrylamide gel electrophoresis(SDS-PAGE) was performed on a Nu-PAGE gel from Novex (San Diego, Calif.)(4-12% MES, running buffer, sample buffer, and/or dithiothreitol, DTTreducing agent). The alum-adjuvanted powder formulations werereconstituted with water and centrifuged to remove the supernatant. Thealum pellet was re-suspended in 200 mM Sodium Phosphate, pH 7 with 0.1%SDS. The liquid suspension was then mixed with sample buffer from theNovex gel kit. The cocktail samples were then heated at 0.95° C. for 5minutes and vortexed prior to loading on the gel. The gels were run for35 minutes at 200V/120 mA/25W using a Novex PowerEase 500 power supply,and then coomassie stained (Novex Colloidal Blue Stain) and destainedwith water. A gel image was scanned on a BioRad gel scanner (ModelGS-700 Imaging Densitometer). The scanner was equipped with quantitationsoftware (Quantity One) that can quantify the intensity of the gelbands. The unit of signal intensity is Optical Density (O.D.). Allsamples were compared against a molecular weight marker (Mark 12,Novex).

The results of both non-reducing and reducing gels are presented in FIG.4. As can be seen, no significant differences between the concentratedliquid sample and the SFD samples were observed, showing that thespray-freeze-drying process of the present invention does not affect theantigen quality.

Example 2 Spray Freeze Drying (“SFD”) of Biopharmaceutical (Protein)Compositions

2.1 Objectives:

-   -   To assess the SFD process for use in preparing biopharmaceutical        (protein pharmaceutical) powders and to further assess the        powdered formulations with respect to the following        criteria: (a) particle (tap density); (b) particle size        distribution; and (c) physical stability of the particles.

A model protein, bovine serum albumin (BSA), was used as the basis forthis study on SFD protein powders produced according to the instantinvention, and the evaluation of these powders based on physicalproperties, including particle size, tap density, and physicalappearance in terms of hygroscopicity and powder flowability. Anotherimportant aspect of the process that was considered was thefreeze-drying condition/cycle. Aggressive drying cycles were attemptedfor two purposes, shortening the drying time and applying higher dryingtemperatures to facilitate particle collapse during drying.

Spray-freeze-drying allows atomised formulation droplets to beimmediately frozen in liquid nitrogen and then freeze dried. The drypowder has a controlled particle size, and the shape of the particle isspherical. This process is highly efficient and has been demonstrated ashaving as benign effect on biopharmaceuticals such as proteins andpeptides. A number of formulation compositions were assessed for theiraffect on resultant particle density, wherein a range of suitableexcipient compositions permits particles of acceptable characteristicsto be prepared from widely different proteins and peptides. A typicalformulation is the combination of an amorphous sugar to provide proteinstability, a crystalline component to render particle strength, and apolymer to offer particle integrity. These excipients must be highlywater soluble to achieve a high solid content in the liquid formulation,a parameter that is essential to producing particles of acceptabledensity. The selection of the particular excipient combination muststrike a balance between hygroscopicity of the resulting powder and theshort-term and long-term physical stability of the biopharmaceuticalguest. Accordingly, these criteria were also applied during theevaluation of the various excipient compositions used in the instantstudy. All of the excipients used in the study are consideredparenterally acceptable and approvable for use in humans.

2.2 Theoretical Considerations of Ultrasonic Atomisation:

Since the atomisation process is an important pre-requisite to attain afairly narrow particle size distribution, some basic theoreticalconsiderations were addressed.

In simple terms ultrasonic atomisation process results frominstabilities created in the liquid capillary waves that form on theatomising surface as a result of the energy provided to the surface inthe form of high frequency vibrations. Drops are then formed when theultrasonic energy exceeds the liquid's surface tension. The sizedistribution of the drops depends principally on the frequency of thenozzle, as well surface tension and specific gravity, although these areminor factors. Equation 2.1 relates the number median diameter to thefrequency, specific gravity and surface tension;d _(N,05)=0.34(8πs/ρf ²)^(1/3)  (2.1)wherein s is the surface tension of the liquid, ρ is the specificgravity and f is the frequency. Thus from Equation 2.1 it can be seenthat frequency is the principal factor in determining drop size as itenters the equation as a square, whilst surface tension and the specificgravity only enter as the first power.

The principal consequence of Equation 2.1 is that the droplet diameteris proportional to f^(2/3). This implies that higher operatingfrequencies produce smaller droplets.

2.3 Spray Freeze Drying (SFD) Apparatus and Methods:

2.3.1 The SFD Apparatus

The SFD apparatus featured an ultrasonic atomizer (Sono-Tek Corporation,Milton, N.Y.) having a spray nozzle (Model #05793) and a power supply(Model #6-05108). The nozzle was equipped with a quasi-electric quartzcrystal capable of vibrating at a specific frequency that determines thesize of the droplets. Ultrasonic nozzles of 60 and 48 kHz frequencieswere used. Circular metal pans (16-cm in diameter by 6-cm in height)were used to contain the liquid nitrogen. For the lyophilization, ashelf freeze dryer (Model #TDS2C2B5200, Dura-Stop, FTS System, StoneRidge, N.Y.) was used. This dryer can hold six metal pans in one batchrun. Other apparatus included a magnetic stirrer with magnetic stirbars, and a peristaltic pump (Model #77120-70, MasterFlex C/L, BarnantCompany, Barrington, Ill.). A schematic of the Spray Freeze DryingProcess is depicted in FIG. 5.

2.3.2 The SFD Methods

The liquid protein formulation was delivered by the peristaltic pump(flow rate of 1-5 mL/min) into the ultrasonic atomiser (48 or 66 kHz).The liquid formulation was then sprayed into a liquid N₂-containing pan.After spraying, the pan containing the frozen particles in liquidnitrogen was transferred to a pre-cooled freeze dryer (−55° C.). Liquidnitrogen will then evaporate. Four sets of freeze-drying conditions thatwere used in this study are listed below in Tables 2.1-2.4. TABLE 2.1Freeze-drying cycle (conservative 48-hour cycle) Stage/Cycle ConditionsFreezing pre-cool shelf temperature (ST) = −50° C. ramp at 2.5° C./minto ST = −55° C., hold for 15 min wait for product temp (PT) = −48° C.,hold for 15 min Primary Drying condenser/vacuum (C/V) switched “on” whencondenser temp. reaches −40° C., vacuum pump turned on wait for chambervacuum to reach 150 mT wait for foreline vacuum to reach 100 mT ramp at1.0° C./min to ST = −10° C., hold for 24 hours Secondary ramp at 1.0°C./min to ST = 20° C., hold for 24 hours Drying

TABLE 2.2 Freeze-drying cycle (semi-aggressive 20-hour cycle)Stage/Cycle Conditions Freezing pre-cool shelf temperature (ST) = −50°C. ramp at 2.5° C./min to ST = −55° C., hold for 15 min wait for producttemp (PT) = −48° C., hold for 15 min Primary Drying condenser/vacuum(C/V) switched “on” when condenser temp. reaches −40° C., vacuum pumpturned on wait for chamber vacuum to reach 150 mT wait for forelinevacuum to reach 100 mT ramp at 1.0° C./min to ST = −10° C., hold for 5hours ramp at 1.0° C./min to ST = 0° C., hold for 5 hours SecondaryDrying ramp at 1.0° C./min to ST = 15° C., hold for 5 hours ramp at 1.0°C./min to ST = 25° C., hold for 5 hours

TABLE 2.3 Freeze-drying cycle (aggressive 20-hour cycle) Stage/CycleConditions Freezing pre-cool shelf temperature (ST) = −50° C. ramp at2.5° C./min to ST = −55° C., hold for 15 min wait for product temp (PT)= −48° C., hold for 15 min Primary Drying condenser/vacuum (C/V)switched “on” when condenser temp. reaches −40° C., vacuum pump turnedon wait for chamber vacuum to reach 150 mT wait for foreline vacuum toreach 100 mT ramp at 1.0° C./min to ST = −10° C., hold for 10 hoursSecondary ramp at 1.0° C./min to ST = 15° C., hold for 5 hours Dryingramp at 1.0° C./min to ST = 25° C., hold for 5 hours

TABLE 2.4 Freeze-drying cycle (most aggressive 16-hour cycle)Stage/Cycle Conditions Freezing pre-cool shelf temperature (ST) = −50°C. ramp at 2.5° C./min to ST = −55° C., hold for 15 min wait for producttemp (PT) = −48° C., hold for 15 min Primary Drying condenser/vacuum(C/V) switched “on” when condenser temp. reaches −40° C., vacuum pumpturned on wait for chamber vacuum to reach 150 mT wait for forelinevacuum to reach 100 mT ramp at 1.0° C./min to ST = −5° C., hold for 6hours Secondary Drying ramp at 1.0° C./min to ST = 25° C., hold for 10hours

After drying, the powder-containing pan was transferred into a dry boxpurged with nitrogen to maintain a relative humidity of <30%.

2.4 Powder Characterization:

2.4.1 Particle Size Analysis

The mean geometric diameter of the particles in the volume distributioncan be determined using an AccuSizer 780 (Particle Sizing Systems, SantaBarbara, Calif.). Based on the light obscuration technique, AccuSizerdetermines the particle size distribution without assuming the shapedistribution of the particle. In addition, the size of the particlepopulation between 10% and 90% (volume) can also be determined. Eachanalysis required approximately 5 mg of the powder sample. Powdersamples are suspended in light mineral oil and sonicated for 5-10seconds to remove agglomeration of particles before analysis.

The mean aerodynamic diameter of the particles in the volumedistribution was determined using a dry powder dispersion-based particlesize analyser (Aerosizer, API). In addition, the size of the particlepopulation between 10% and 90% (in volume distribution) was alsoreported for each particle size distribution. Each analysis requiredapproximately 5 mg of the powder sample.

2.4.2 Image Analysis

Visual analysis of the particles was performed using an opticalmicroscope (Model DMR, Leica, Germany) with 10×-eyepiece lens and10×-objective lens. The system was equipped with a Polaroid camerasystem for image output. Digital images were captured and stored.

2.4.3 Tap Density

Each powder sample was weighed in a glass vial and gently tapped againstthe lab bench for 20 times. By visual inspection, water of an equivalentvolume to that of the powder was placed into an empty vial of the sametype. The tap density of the powder sample could be calculated bydividing the powder sample weight with the water sample weight (assumingwater density=1 g/mL).

2.4.4 Moisture Content Analysis

Approximately 4-5 mg of the powder sample was recovered from thetrilaminate cassettes and transferred into an aluminum weighing-vesseland the weight was recorded. The sample was loaded into a Karl FisherCoulometer (Model 737, Brinkmann) equipped with a drying oven (Model707). The sample was heated to 150° C. for 150 sec with a gas flow rateof 100 mL/min within the drying oven. Sample extraction time was 120seconds.

2.5 Pharmaceutical Formulations and Results:

In the series of experiments discussed herein below, variousformulations were tested wherein two excipients were kept constant,namely mannitol and trehalose. Trehalose was selected primarily forstabilizing the protein during lyophilization and long-term storage.Mannitol was added to the formulation to impart rigidity to theparticles, as mannitol is a crystalline material.

The powder formulations that were produced using the SFD process of thepresent invention and the particular apparatus and methods describedabove. In the first series of experiments, formulations containingpolyvinylpyrrolidone (PVP) were assessed. PVP is a parenterallyacceptable excipient and imparts plasticity to the formulation, andhence was selected as a preferred bulking agent.

2.5.1 PVP Formulations

In the following series of experiments, the objective was to determinethe particle physical properties when the concentration of the PVPbulking agent was altered from 18 to 36% w/w. The particularformulations tested are reported below in Table 2.5. TABLE 2.5 SFD PVPformulations Solids Nozzle Freeze Batch Formulation Composition ContentFreq. Drying Number (% w/w) (% w/w) (kHz) Cycle 156-16-1 10% BSA, 45%trehalose, 35 60 See Table 27% mannitol, 18% PVP 2.1 (K17) and 0.1%Pluronic F68. 156-16-2 10% BSA, 44.9% 35 48 See Table trehalose, 26.9%mannitol, 2.1 18% PVP (K17), 0.1% methionine and 0.1% Pluronic F68156-16-3 10% BSA, 26.9% 35 60 See Table trehalose, 26.9% mannitol, 2.135.9% PVP (K17), 0.1% methionine and 0.1% Pluronic F68. 156-16-4 10%BSA, 26.9% 35 48 See Table trehalose, 26.9% mannitol, 2.1 35.9% PVP(K17), 0.1% methionine and 0.1% Pluronic F68.

Image Analysis Results:

Photomicrographs (FIGS. 6 a-6 d) of the SFD formulations defined inTable 2.5 reveal that the particles are of a spherical morphology, witha fairly uniform particle size. There are some particles that areagglomerated, which is a consequence of PVP present in the formulations.PVP is an effective binder, and formulations containing this excipientwill have some tendency to agglomerate due to the adhesive nature of thepolymeric excipient.

Particle Size Results:

As seen below in Table 2.6, the mean particle sizes of the PVPformulations were similar, with the exception of batch number 156-16-3,which had a smaller mean particle size. Comparison of batches 156-16-1and 156-16-3, which were both manufactured using a 60 kHz ultrasonicnozzle (the only difference between the two formulations being the ratioof the excipients), reveals that the 3:3:4 excipient ratio producedsmaller particles. Similarly, comparison of batches 156-16-2 and 156-164showed the same trend, although the effect was less marked. TABLE 2.6Particle size results Mean Size Median size Batch number (μm)D_(0.10)-D_(0.90) (μm) 156-16-1 39.2 ± 1.4 24.9-59.3 40.0 156-16-2 40.4± 1.4 26.8-60.3 40.8 156-16-3 36.3 ± 1.3 24.7-52.7 36.6 156-16-4 39.6 ±1.4 26.3-58.9 39.9

Moisture Content Analysis:

The Karl Fischer results presented in Table 2.7, below, reveal that themoisture content of all of the PVP formulations was <3%. These resultsfurther indicate that the ratio of excipients 3:3:4 produced a drierproduct. TABLE 2.7 Karl Fischer (moisture content) results Batch Number% Moisture 156-16-1 2.9 156-16-2 2.9 156-16-3 1.9 156-16-4 2.4

Particle Density Results:

As can be seen by the results reported in Table 2.8 below, the tapdensities of all four PVP formulations were similar and withinacceptable ranges. TABLE 2.8 Tap density Batch Number Tap Density(g/cm³) 156-16-1 0.65 156-16-2 0.66 156-16-3 0.64 156-16-4 0.67

2.5.2 Various Sugar Formulations

In the following series of experiments, the objective was to determinethe particle physical properties using differing combinations of sugars(raffinose, sucrose) and other common excipients (glycine). Theparticular formulations tested are reported below in Table 2.9. TABLE2.9 SFD sugar formulations Ultrasonic Formulation Solids Nozzle FreezeBatch Composition Content frequency Drying Number (% w/w) (% w/w) (kHz)Cycle 156-35-1 10% BSA, 36% raffinose, 35 60 See 27% trehalose, 27%Table mannitol. 2.2 156-35-2 10% BSA, 36% raffinose, 35 60 See 36%mannitol and 18% Table PVP (K17). 2.2 156-35-3 30% BSA, 40% raffinose 3560 See and 30% mannitol. Table 2.2 156-42-1 10% BSA, 36% raffinose, 3560 See 27% trehalose, 27% Table mannitol. 2.3 156-42-2 10% BSA, 27%raffinose, 35 60 See 27% mannitol 18% Table glycine and 18% 2.3trehalose. 156-42-4 10% BSA, 27% raffinose, 35 60 See 27% sucrose and36% Table mannitol. 2.3

Image Analysis Results:

As can be seen in FIGS. 7 a-7 f, the particles prepared by the SFDprocess from the formulations defined in Table 2.9 have a sphericalmorphology, with a fairly uniform particle size. However, batch numbers156-35-1, 156-35-2 and 156-35-3 (FIGS. 7 a, 7 b and 7 c, respectively)appear to have a few oversize particles. Batch number 15642-2 (FIG. 7 e)had an irregular morphology and appears to be in the process ofdeliquescence, suggesting the highly hygroscopic nature of theformulation.

Particle Size Results:

The particle size results of the formulations of Table 2.9 are reportedbelow in Table 2.10 and generally correspond to the estimated sizesobtained from the photomicrographs. TABLE 2.10 Particle Size ResultsBatch Mean Size Median size Number (μm) D_(0.10)-D_(0.90) (μm) 156-35-139.5 ± 1.3 27.4-55.3 40.1 156-35-2 38.9 ± 1.3 26.0-55.7 39.8 156-35-334.4 ± 1.4 22.7-50.0 35.3 156-42-1 34.4 ± 1.3 23.3-49.5 35.1 156-42-236.1 ± 1.3 26.4-48.8 36.4 156-42-4 38.8 ± 1.3 27.4-52.9 40.0

Moisture Content Analysis:

The Karl Fischer results presented in Table 2.11, below, reveal that themoisture content of all of the tested formulations was <5%. As can beseen, batch number 156-35-2 had the lowest residual moisture, likely dueto the increased amount of mannitol in that formulation. TABLE 2.11 KarlFischer (moisture content) results Batch Number % Moisture 156-35-1 4.8156-35-2 3.0 156-35-3 4.2

Particle Density Results:

As can be seen by the results reported in Table 2.12, below, the tapdensities of all of the tested formulations were similar and withinacceptable ranges. As can also be seen, the addition of glycine toformulation for batch number 156-42-2 caused a marked increase in tapdensity relative to the other formulations. The variables that can altertap density of a powder are multifaceted and depend principally on theparticle size, particle size distribution, crystal habit and rugosity.Alterations in these variables by introduction of another excipient oran increase in the amount of excipient/active formulation will lead topredictable differences in particle tap densities.

The use of raffinose as a bulking agent in these formulations resultedin particles of acceptable physical characteristics, and there was alower incidence of agglomeration when compared to the PVP formulations.TABLE 2.11 Tap density Batch Number Tap Density (g/cm³) 156-35-1 0.65156-35-2 0.57 156-35-3 0.59 156-42-1 0.68 156-42-2 0.75 156-42-4 0.67

2.5.3 Various Dextran Formulations

In the following series of experiments, the objective was to determinethe particle physical properties using dextran as the bulking agent.Dextran forms a glass having a high glass transition temperature (TG).The particular dextran-containing formulations tested are reported belowin Table 2.13. TABLE 2.13 SFD dextran formulations Solids Nozzle FreezeFormulation Composition Content frequency Drying Batch Number (% w/w) (%w/w) (kHz) Cycle 156-35-4 10% BSA, 27% trehalose, 35 60 See 27% mannitoland 36% Table dextran (10 kDa). 2.2 156-42-3-1 10% BSA, 27% trehalose,35 60 See 27% mannitol and 36% Table dextran (10 kDa). 2.3 156-42-3-210% BSA, 27% trehalose, 35 60 See 27% mannitol and 36% Table dextran (10kDa). 2.3 156-53-1 10% BSA, 27% trehalose, 35 60 See 27% mannitol and36% Table dextran (10 kDa). 2.4 156-61-1 10% BSA, 27% trehalose, 35 60See 27% mannitol and 36% Table dextran (10 kDa). 2.4 156-65-1 10% BSA,36% trehalose, 40 60 See 18% mannitol, 18% arginine Table glutamate, 18%dextran (10 kDa). 2.4

Image Analysis Results:

As can be seen in FIGS. 8 a-8 f, the SFD dextran formulations (definedin Table 2.13) provided particles with a spherical morphology, a narrowsize distribution and there was also a noticeable lack of agglomeratedparticles.

Particle Size Results:

The particle size results from the assessment of the various dextranbatches (the formulations of Table 2.13) are reported herein below inTable 2.14, and generally correspond to the estimated sizes obtainedfrom the photomicrographs. As can be seen, there was a marked increasein the mean particle size of batch number 156-53-1 when compared tobatch number 156-42-3-1, but this can be explained by the PSD generatedby the Aerosizer, which was skewed more to the right, when compared withthe PSD of batch number 156-42-3-1, which is indicative of largeparticles. Batch number 156-61-1, which is a scaled-up formulation ofbatch number 156-53-1 yielded a similar PSD. Batches numbers 156-53-1and 15642-3-1 have similar BSA and excipient compositions, however afreeze-drying cycle of 16 hours was utilised for the former.

These Aerosizer data also reveal that there was a marked increase in themean particle size when using a shorter drying cycle, however inspectionof FIGS. 8 c and 8 f does not show any significant differences in termsof morphology or size. From these data it can be postulated that alonger drying cycle results in a more collapsed particle and this mayhave an effect on the intra-particle porosity, in which case this wouldaffect the particle size as measured by the Aerosizer as the aerodynamicsize depends on intraparticle porosity. Batch number 156-65-1 had thelargest mean particle size. This can be attributed to the increase insolids content relative to the other dextran formulations of Table 2.13.TABLE 2.124 Particle size results Mean Size Median size Batch number(μm) D_(0.10)-D_(0.90) (μm) 156-35-4 33.1 ± 1.3 23.3-46.1 33.6156-42-3-1 35.0 ± 1.3 24.7-49.1 35.4 156-42-3-2 33.6 ± 1.3 23.4-47.334.2 156-53-1 39.2 ± 1.4 26.2-57.6 39.9 156-61-1 39.7 ± 1.4 25.4-61.940.1 156-65-1 45.3 ± 1.3 30.8-64.9 46.5Particle Density Results:

As can be seen by the results reported in Table 2.15, below, the tapdensities of all of the tested dextran formulations were similar andwithin acceptable ranges. A review of these densities also indicatesthat the freeze-drying time had a negligible overall effect on the tapdensity.

The marked increase in the mean particle size of batch number 156-53-1,when compared to batch number 156-42-3-1, can not be due to theproduction of a more collapsed particle as the tap densities and lightmicroscopy photomicrographs would have indicated a difference betweenthe two batches, hence the difference between the mean particle sizes ofthe two batches is attributed to sampling variability. TABLE 2.13 Tapdensity Batch Number Tapped Density (g/cm³) 156-35-4 0.54 156-42-3-10.57 156-42-3-2 0.56 156-53-1 0.53 156-61-1 0.56 156-65-1 0.61

2.5.4 Various Salt Formulations

In the following series of experiments, the objective was to determinethe particle physical properties of formulations incorporating differingcombinations of a salt bulking agent (arginine glutamate) and othercommon excipients (alanine, Pluronic, methionine). The particularformulations tested are reported below in Table 2.16. TABLE 2.146 SFDsalt formulations Nozzle Freeze Batch Formulation Composition SolidsFrequency drying Number (% w/w) Content (kHz) Cycle 156-57-1 10% BSA,36% trehalose, 35 60 See 36% mannitol, 18% Table 2.3 alanine. 156-57-210% BSA, 27% trehalose, 35 60 See 27% mannitol 36% Table 2.3 arginineglutamate. 156-65-2 10% BSA, 36% trehalose, 40 60 See 18% mannitol, 36%Table 2.4 arginine glutamate. 156-71-1 10% BSA, 36% trehalose, 40 60 See18% mannitol, 36% Table 2.1 arginine glutamate. 156-76-1 10% BSA, 35.9%trehalose, 35 60 See 18% mannitol, 35.9% Table 2.4 arginine glutamate,0.1% Pluronic F168 and 0.1% methionine. 156-76-2 10% BSA, 26.9%trehalose, 35 48 See 26.9% mannitol, 35.9% Table 2.4 arginine glutamate,0.1% Pluronic F168 and 0.1% methionine.

Image Analysis Results:

As can be seen in FIGS. 9 a-9 f, the SFD salt formulations defined inTable 2.16, provided particles with a spherical morphology and narrowsize distribution. FIG. 9 a is a photomicrograph of batch number156-57-1, which shows that the particles have agglomerated after thefreeze drying process, moreover the particles shown in FIG. 9 a have athick rounded edge, which is indicative of deliquescence. The other saltformulations do not show any evidence of agglomeration.

Accordingly, X-ray powder diffraction (XRPD) was conducted on thearginine glutamate and a spray freeze dried formulation containing theaforementioned excipient. This was conducted to elucidate the morphologyof the excipient prior to freeze drying and after freeze drying.Analysis of the XRPD pattern for arginine glutamate prior to freezedrying showed distinct peaks, which is indicative of a crystallinematerial. Analysis of the XRPD pattern for batch number 156-76-1 showeda diffuse halo, suggesting an amorphous formulation.

Particle Size Results:

The particle size results of the various salt formulations (theformulations of Table 2.16) are reported herein below in Table 2.17, andgenerally correspond to the estimated sizes obtained from thephotomicrographs. TABLE 2.15 Particle size results Mean Size Median sizeBatch number (μm) D_(0.10)-D_(0.90) (μm) 156-57-1 40.3 ± 1.3 27.8-57.640.8 156-57-2 37.7 ± 1.4 23.7-58.0 38.3 156-65-2 44.1 ± 1.4 28.2-66.145.4 156-71-1 41.3 ± 1.4 26.0-63.5 42.7Particle Density Results:

As can be seen by the results reported in Table 2.18, below, the tapdensities of all of the tested formulations were relatively high, withthe single exception of batch number 156-76-1, and all within acceptableranges. These results demonstrate that the use of arginine glutamate asthe bulking agent in the formulations of the present invention providesparticles of acceptable physical characteristics, however, the use ofalanine was not deemed optimal due the deliquescence of the formulation.TABLE 2.16 Tap density Batch Number Tapped Density (g/cm³) 156-57-1 0.67156-57-2 0.69 156-65-2 0.63 156-71-1 0.66 156-76-1 0.46 156-76-2 0.57

2.5.5 Further Salt Formulations

As with the above series of experiments, the objective of this series ofexperiments was to determine the particle physical properties offormulations incorporating combinations of different alternative bulkingagents (arginine aspartate) and other common excipients (Pluronic F168,methionine, Tween 80). The particular formulations tested are reportedbelow in Table 2.19. TABLE 2.179 SFD formulations Nozzle Freeze BatchFormulation Composition Solids frequency drying Number (% w/w) Content(kHz) cycle 156-80-1 10% BSA, 27% trehalose, 35 60 See Table 27%mannitol, 36% 2.4 arginine aspartate. 156-80-2 10% BSA, 5% Pluronic 3560 See Table F168, 59.5% trehalose and 2.4 25.5% mannitol. 156-80-3 10%BSA, 35.9% 40 60 See Table trehalose, 18% mannitol, 2.4 35.9% arginineglutamate, 0.1% methionine and 0.1% Tween 80.

Image Analysis Results:

As can be seen in FIGS. 10 a-10 c, the SFD salt formulations defined inTable 2.19 produced particles with a spherical morphology. However, anumber of oversize particles are evident, particularly in batch number156-80-3. In addition, batch number 0.156-80-2 (FIG. 10 b) had particlesthat seemed to be fused together, likely as a consequence of the highPluronic content in the formulation.

Particle Size Results:

The particle size results of the various salt formulations (theformulations of Table 2.19) are reported herein below in Table 2.20, andgenerally correspond to the estimated sizes obtained from thephotomicrographs depicted in FIGS. 10 a-10 c. Batch number 156-80-1yielded the smallest mean particle size, due to the lower solids contentused, whilst batch number 156-80-2 yielded the largest particle sizewhich was partially as a consequence of particle agglomeration/fusion(see. FIG. 10 b). TABLE 2.20 Particle size results Mean Size Median sizeBatch number (μm) D_(0.10)-D_(0.90) (μm) 156-80-1 33.6 ± 1.4 22.5-48.434.6 156-80-2 41.8 ± 1.4 26.2-63.2 43.4 156-80-3 37.7 ± 1.4 24.9-55.938.6

Particle Density Results:

As can be seen by the results reported in Table 2.21, below, the tapdensities of all of the tested formulations were relatively high andwithin acceptable ranges. The formulation of batch number 156-80-1 had arelatively lower density due to a lower starting solids content (35%).These results demonstrate that the use of arginine aspartate as thebulking agent in the formulations of the present invention providesparticles of acceptable physical characteristics. TABLE 2.21 Tap densityBatch Number Tapped Density (g/cm³) 156-80-1 0.51 156-80-2 0.72 156-80-30.63

Example 3 Spray Freeze Drying of Alum-Adjuvanted Vaccine Compositions

3.1 Objectives:

-   -   To assess the SFD process for use in preparing alum-adjuvanted        vaccine powders and to further assess the powdered formulations        with respect to their in vivo performance using epidermal powder        immunization (“EPI”) and conventional needle and syringe        administration techniques. Hepatitis B vaccine (Alum-HBsAg) and        a diphtheria/tetanus toxoid vaccine (Alum-DT) were selected for        the studies since the Alum-HBsAg composition contains aluminium        hydroxide adjuvant and the Alum-DT composition contains        aluminium phosphate adjuvant.        3.2 Materials:

The chemicals and excipients that were used to produce the variousvaccine compositions used in this study are summarized in Table 3.1below. All alum formulations were concentrated by centrifugation(Allergra 6R Centrifuge, Beckman Instrument, Palo Alto, Calif.) prior touse. TABLE 3.1 Chemicals/excipients used in the study. Chemical Lot #Source Comment Aluminum 8934 Accurate Chemical Manufactured by HCIphosphate (Adjus- and Scientific Biosector Phos, 2% AlPO₄) (Westbury,NJ) (Frederikssund, Denmark) Aluminum Accurate Chemical Manufactured byhydroxide and Scientific Superflos Biosector (Alhydrogel, 3% (Vedbaek,Denmark) Al(OH)₃) Diphtheria toxoid G9334 Accurate Chemical Manufacturedby (dT, MW 58 kDa) and Scientific Statens Serum Institute, Denmark, andprovided at 5 mg/mL (1 Lf = 2.42 μg), used as supplied. Tetanus toxoid(tT, G9486 Accurate Chemical Manufactured by MW 150 kDa) and ScientificStatens Serum Institute, Denmark, and provided at 2 mg/mL (1 Lf = 2.44μg), used as supplied. Alum phosphate- CSL Limited Bulk containing 5 w/v% adjuvanted DT (Parkville, alum phosphate Australia) adsorbed with bothdT and tT at 563 Lf/mL Alum hydroxide- Rhein Amaericana 20 μg HBsAgadsorbed adjuvanted hepatitis- S.A. (Buenos Ares, to 0.5 mg of aluminumB surface antigen Argentina) or 1.5-mg of aluminum (HBsAg) hydroxide.Dextran (MW 18H0568 Sigma (St. Louis, MO) Reagent grade, used as 37,500Da) supplied Glycine 28H0103 Sigma Reagent grade, used as suppliedMannitol 127H0960 Sigma Reagent grade, used as supplied Trehalosedihydrate 28H3797 Sigma Reagent grade, used as supplied3.3 Methods:

3.3.1 Spray-Freezing (SF) and Spray-Freeze-Drying (SFD)

Liquid formulations were delivered by a peristaltic pump (Model#77120-70, MasterFlex C/L, Barnant Company, Barrington, Ill.) at a flowrate of 2.0 mL/min into an ultrasonic atomizing system (Sono-TekCorporation, Milton, N.Y.) consisting of a spray nozzle (Model #05793)and a power supply (Model #06-05108). The nozzle is equipped with aquasi-electric quartz crystal capable of vibrating at a specificfrequency that determines the size of the droplets. A 60 kHz sprayingnozzle produces droplets mostly within the range of 20-80 μm. Atomizeddroplets were sprayed into a liquid N₂-containing pan (16 cm in diameterby 6 cm in height). For formulations subjected to thespray-freezing/thawing experiment, the frozen powder was transferred toa glass vial and thawed at ambient conditions. For frozen dropletsundergoing drying, the pan containing frozen particles in liquidnitrogen was transferred to a pre-cooled (−55° C.) shelf freeze dryer(Model #TDS2C2B5200, Dura-Stop, FTS System, Stone Ridge, N.Y.). Theliquid nitrogen evaporated in a few minutes. The freeze-drying conditionwas set at −25° C. for 18 hours and 20° C. for 10 hours. The rampingrate was 1° C./minute consistently. The vacuum pressure was 100 mTthroughout the cycle. After drying, the powder-containing pans weretransferred into a dry box purged with nitrogen (at <30% relativehumidity) for powder collection. The same lyophilization cycle was usedfor liquid formulations without SF with freezing achieved by storing ina −20° C. freezer overnight.

3.3.2 Powder Formation by Compress/Grind/Sieve (C/G/S) Method

To prepare powders of high density for the epidermal powder immunization(EPI) study, freeze-dried (FD) and SFD formulations were compressed in astainless steel dye of 13-mm in diameter (Carver Press, Wabash, Ind.) ata pressure of 12,000-15,000 pounds for 5-10 minutes. The compresseddiscs were ground manually using a mortar and pestle, and then theground powder was manually sieved through a stack of 3-in sieves (FisherScientific Products, Pittsburgh, Pa.) of four sizes, 20, 38, 53, and 75μm.

3.3.4 Spray-Drying (SD)

A bench-top mini spray dryer (Buchi B-191, Brinkmann, Westbury, N.Y.)was used to prepare placebo alum formulations. Using compressed air froman in-house supply (˜80 psi), a two-fluid nozzle (0.5 mm) atomized theaqueous feed solution. The standard operating conditions were: inlet airtemperature of 130° C., drying air blown at the full scale, atomizingair flow rate of 500 L/hr, and liquid feed rate of 10 mL/min. Thiscondition resulted in an outlet air temperature of 70° C.

A laboratory spray dryer (Mobile Minor, Niro A/S, Soeborg, Denmark) wasused to prepare Alum-DT formulation with the following conditions. Thetwo-fluid nozzle was operated at an atomizing pressure of 2 bar. Theinlet air temperature was set at 160° C. drying air with full-blowndrying air. As the liquid was fed at 30 mL/min, the air outlettemperature measured at 65-70° C.

3.3.5 Air-Drying (AD)

Liquid alum-adjuvanted vaccine formulations were placed in a polystyreneweigh boat and allowed to dry overnight under the ambient conditions.Gentle agitation by a magnetic bar stirring was applied throughout theprocess to minimize phase separation.

3.3.6 Optical Microscopy

Visual analysis of the particles was performed using an opticalmicroscope (Model DMR, Leica, Germany) with 10×-eyepiece lens and10×-objective lens. The system was equipped with a Polaroid camerasystem for image output.

3.3.7 Particle Size Analysis

The mean geometric/aerodynamic diameter of the particles in the volumedistribution was determined using a time-of-flight particle sizeanalyzer (Aerosizer, API, Minneapolis, Minn.). The mean volumetric sizewas calculated by the software using the density of 1.0 and particlepopulation between 10% (D₁₀) and 90% (D₉₀) was reported for particlesize distribution. Each analysis requires approximately 3-5 mg of thepowder sample. For liquid suspensions, the particle size distributionwas measured using a light obscuration-based particle size analyzer(AccuSizer 780, Particle Sizing Systems, Santa Barbara, Calif.).

3.3.8 SDS-PAGE

Coomassie colloidal-stained SDS-polyacrylamide gel electrophoresis(SDS-PAGE) was performed on a Nu-PAGE gel from Novex (San Diego, Calif.)(4-12% MES, running buffer, sample buffer, and/or Dithiothreitolreducing agent). The alum-adjuvanted powder vaccine formulations werereconstituted with water and centrifuged to remove the supernatant. Thealum pellet was re-suspended in 200 mM sodium phosphate, pH 7 with 0.1%SDS. The liquid suspension was then mixed with sample buffer from theNovex gel kit. The cocktail samples were then heated at 95° C. for 5minutes and vortexed prior to loading on the gel. The gels were run for35 minutes at 200V/120 mA/25W using a power supply (PowerEase 500,Novex), and then coomassie stained (Novex Colloidal Blue Stain) anddestained with water. The gel images were scanned on a gel scanner(Model GS-700 Imaging Densitometer, BioRad) equipped with a quantitationsoftware (Quantity One), which can quantify the intensity of the gelbands. The unit of signal intensity is Optical Density (O.D.). Allsamples were compared against a molecular weight marker (Mark 12,Novex).

3.3.9 EPI Using a PowderJect® Powder Injection Device

A PowderJect® powder injection device (needleless syringe) was used toimmunize hairless guinea pigs. The device is approximately 15 cm inlength and includes a gas cylinder (5-ml volume), rupture chamber, atrilaminate particle cassette, a nozzle, and a silencer element. Thestainless steel gas cylinder is filled with medical grade helium gas to40-bar pressure. The trilaminate cassette (11-mm O.D., 6-mm I.D., and4-mm height) is constructed of a thick ethylene vinyl acetate washerwith rupture membranes heat sealed to each side within which thepowdered vaccine sample is housed. The rupture membranes are formed froma thin film (20 μm) made of semi-transparent polycarbonate. Uponactuation, the helium gas is released from the gas cylinder and causespressure build-up in the rupture chamber. The escaping gas overcomes therupture strength of the rupture membranes, causing the membranes torupture, whereby the gas sweeps through the trilaminate cassette andpropels the vaccine powder as projectiles into the skin. The helium gasis reflected off the skin and exhausted through the silencer element.The depth of powder penetration was experimentally optimized to deliverpowders to the epidermal layer of the skin for optimal tolerance andmaximal efficacy.

3.3.10 Mice Immunization and Serum Collection

Five to seven week-old female BALB/c mice (Harlen-Sprague-Dawley,Indianapolis, Ind.) were used to assess the immunogenicity of powderedalum-adsorbed hepatitis B vaccines. FD and SFD powder formulations werereconstituted with distilled water and administered by intraperitoneal(IP) injection using a 26⅕ needle. Each injection administered 200 μl ofsolution containing 2 μg of hepatitis B surface antigen adsorbed onalum. Control mice were immunized with the same dose of untreated liquidhepatitis B vaccine. A boost immunization was administered on day 28.

Blood was collected via retro-orbital bleeding under anaesthesia priorto each vaccination and two weeks post boost.

3.3.1.1 Guinea Pig Immunization and Serum Collection

Hairless guinea pigs (Charles River, Wilmington, Mass.) were used toassess the immunogenicity of powder formulations of alum adsorbeddiphtheria toxoid (dT) and tetanus toxoid (tT) following EPI. Thegeneral methods for EPI are described in detail herein above and in theart. Briefly, one mg of the powdered vaccine compositions being testedwas dispensed into a trilaminate cassette. The cassette was insertedinto the PowderJect powder injection device at the time of immunization.The device was placed against the left inguinal skin of the animals andactuated by releasing the compressed helium at 40-bar pressure from thegas cylinder. Control animals were immunized with 0.20 mL of DT vaccinein saline by intramuscular (IM) injection using a 26½-gauge needle.

Blood was collected via the kerotid blood vessel prior to eachvaccination and two weeks post boost.

3.3.12 ELISA

The antibody responses to diptheria toxoid (dT) and tetanus toxoid (tT)components of the Alum-DT vaccine and to the HBsAg antigen component ofthe Alum-HBsAg vaccine were determined using a modified ELISA method. A96-well plate (Costar, Fisher Scientific Products, Pittsburgh, Pa.) wascoated with 0.1 μg of antigen (HBsAg, dT, or tT) in 30 mM phosphatebuffered saline (PBS), pH 7.4, per well overnight at 4° C. Plates werewashed 3 times with tris-buffered saline (TBS), pH 7.4, containing 0.1%Brij-35, and incubated with test sera diluted in PBS containing 5% drymilk for 1.5 hr. A standard serum, containing a known level ofantibodies to dT, tT, or HBsAg, was added to each plate and used tostandardize the titer in the final data analysis. The plates were thenwashed and incubated with biotin-labeled goat anti-mouse antibodies(1:8,000 in PBS, Southern Biotechnology. Associate, Birmingham, Ala.)for 1 hr at room temperature. Finally, the plates were washed anddeveloped with TMB substrate (Bio-Rad Laboratories, Melville, N.Y.). Theendpoint titers of the sera were determined by 4-parameter analysisusing the Softmax Pro 4.1 program (Molecular Devices, Sunnyvale, Calif.)and defined as the reciprocal of the highest serum dilution with an ODreading above the background by 0.1. A reference serum with apre-determined titer was used on every plate to calibrate the titers andadjust assay-to-assay and plate-to-plate variation.

3.4 Powder Characterization:

Current commercial alum-adjuvanted vaccines are formulated atapproximately 2 w/v % of aluminum salt in saline for injection.Accordingly, commercially available vaccine compositions were reviewedbefore and after freezing using an optical microscope. The results ofthe study are depicted in FIGS. 11 a-11 d. Based on optical microscopy,the Adju-Phos adjuvant (2 w/v % placebo AlPO₄ gel) shows smooth andsandy texture without discernible particles. After freezing at −20° C.,the thawing gel develops immediately significant coagulation (see FIG.11 a), but the same gel shows only slight aggregation (light dots) afterspray-freezing (see FIG. 11 b). The same difference was observed for theAlhydrogel adjuvant (3 w/v % placebo Al(OH)₃) after freezing at −20° C.(see FIG. 11 c) and spray-freezing (see the dark particles in FIG. 11d). By appearance, large particles were visible in the gel solution thathad been frozen in the −20° C. freezer and they rapidly settled to thebottom of the container. In addition, the volume of the settled alumparticles was significantly greater than that of the starting gel. Allthese observations suggest that alum gels will coagulate after regularfreezing. Coagulated alum gels are not reversible even under mechanicalforce such as sonication or vortexing. On the other hand, the absence ofcoagulation with the spray-frozen gel confirms that extremely fastfreezing significantly reduces the coagulation tendency of alum gelregardless of its salt type even in the absence of bulking orstabilizing agents. After lyophilization of the frozen gels, the extentof coagulation of the reconstituted alum appears to be similar to thefreeze/thaw samples, suggesting that freezing is a primary cause of alumcoagulation.

3.5 In Vivo Performance:

3.5.1 Effect of Alum Coagulation on Immunogenicity of Alum-HBsAg

A mouse model was used to test if the immunogenicity of the Alum-HBsAgvaccine composition would be affected by the drying methodology and thesize of the coagulated particles. Table 3.2, below, summarizes thestudy. TABLE 3.2 Immunogenicity Study for Alum-HBsAg powderformulations. Drying Process Group (n = 8) Formulation (particle size) 1Q FD 2 Q FD (<20 μm) 3 Q FD (38-53 μm) 4 Q FD (53-75 μm) 5 Q SFD (38-53μm) 6 R SFD (38-53 μm) 7 Control not dried (liquid)

The formulations used in the study were as follows. Formulation Q: Alumhydroxide (3.0 w/v %)/mannitol (1.9 w/v %)/glycine (0.5 w/v %)/dextran(0.61 w/v %) in which the alum concentration was achieved by combiningthe Alum-HBsAg with the placebo alum hydroxide gel. Formulation R: Alumhydroxide (0.6 w/v %)/mannitol (2.8 w/v %)/glycine (1.2 w/v %)/dextran(0.58 w/v %). Control: the commercial Alum-HBsAg product (Rhein Biotech)used as supplied by the manufacturer.

The SFD powders prepared from Formulations Q and R differ in alum saltconcentration. Powders for Groups #24 were prepared by compressing theFD Formulation Q followed by grinding and then sieving to produce 3different particle size-fractions. All powdered compositions were fullycharacterized as described above in Examples 1 and 2. The opticalmicrographs of the reconstituted powders again suggested that the SFDpowder could be readily re-suspended in water while the FD formulationwas highly coagulated. Another characterization method involvedre-suspending the powder sample in water and subjecting tolight-scattering particle size analysis (AccuSizer). The results of theparticle size analysis are depicted in FIGS. 12 a and 12 b. FIG. 12 ashows the alum particle size distribution for the two SFD powdersfalling in the same particle size range as the starting gel, suggestingno detectable aggregated particles. However, the reconstitutedFD/compress/grind/sieve powder (3845 μm, Group #3 in Table 3.2) shows aparticle size range of 5-50 μm with a peak at 45 μm (FIG. 12 b), whichoverlaps with the size of dry particles before rehydration.

In order to assess stability of the Alum-HBsAg compositions upon drying,SDS-PAGE analysis was performed under both non-reducing and reducingconditions. The results from the optical density scans of thenon-reduced SDS-PAGE analysis are reported below in Table 3.3. TABLE 3.3Optical density of HBsAg band (non-reduced SDS-PAGE) Light IntensityFormulation (O.D.) Formulation Q, FD 847 Formulation Q FD (particle 1235size <20 μm) Formulation Q, FD (particle 1021 size 53-75 μm) FormulationQ, SFD 1507 (particle size 38-53 μm) Formulation R, SFD 1479 (particlesize 38-53 μm) Control (liquid) 1671

In its native state, HBsAg is a highly aggregated particle of 22 nm indiameter. No formulations dissociated into monomeric form under thenon-reducing condition. However, the light intensity of the single bandat the top of the SDS-PAGE gel differs among the formulations (Table3.3). For example, the light intensity for the two SFD formulations isslightly lower than that for the control (starting liquid formulation).The FD formulation showed the lightest band while band intensityincreased when the FD formulation was formulated into particles usingC/G/S. Interestingly, band intensity increased with decreasing particlesize. This observation appears to be related to alum gel coagulationsince the antigen desorption from the surface of aggregated alumparticles may be restricted or blocked. Grinding the FD alum gelgenerated new surfaces exposing more antigen. The powder's specificsurface area increases as the particle size decreases. Under reducingconditions, HBsAg particles were reduced to monomers of approximately 24kDa. There was no difference observed in band pattern and intensityamong all the formulations. This is probably due to the ease with whichmonomeric HBsAg can diffuse out of the tightly packed alum aggregates.

Immunogenicity of the various Alum-HBsAg formulations described in Table3.2 above was tested in the mouse model. The dose of HBsAg administeredto each animal was 2 μg per I-mg powder that was reconstituted in waterand delivered by IP injection. Serum samples were collected 4 weeksafter prime and two weeks after boost. Serum antibodies were determinedusing the standard ELISA and the results are summarized in FIG. 13.

As can be seen in FIG. 13, compared to the untreated liquid vaccine(Control), the FD HBsAg vaccine composition (Group 1) showed diminishedimmunogenicity. In addition, the particle size of the alum-containingpowder had a pronounced effect on immunogenicity of Alum-HBsAg. Theimmunogenicity of the freeze-dried formulations had an inversecorrelation with the size of the particles (Groups 2, 3, and 4). Thelarger particle size fractions were less immunogenic than the smallerparticle size fraction. This is consistent with the SDS-PAGE result andmight be explained by the availability of HBsAg from the coagulated alummatrix. Smaller particles have a greater specific surface area, therebyallowing more HBsAg to be released from the alum matrix in vivo. Analternative explanation is that large coagulated particles are too bigto be phagocytosed by antigen presenting cells, thus, the adsorbedvaccine antigen (HBsAg) is not available to the immune system.Regardless of the mechanism, this data clearly indicated that large sizeparticles associated with coagulation also correlated with the loss ofvaccine potency.

The SFD formulations (Q and R) elicited a significantly higher antibodyresponse than the FD counterparts. This result confirms that alumcoagulation caused an immunogenicity loss of HBsAg and that the fastfreezing rate by SFD is an effective approach to preserving the alumadjuvant activity. The effect of alum concentration in the SFD powderformulation on immunogenicity is important. Although no clearlydetectable coagulation was seen with SFD formulation Q, which contained3.0 w/v % of Alum HBsAg, this formulation induced an antibody titer thatwas approximately 1-log of magnitude lower than the IM injectioncontrol. The SFD formulation with 0.6% alum content had no coagulationand induced an antibody titer that was indistinguishable from the IMinjected animals (p>0.05, Student test). This result suggests thatlowering alum concentration and fast freezing are the most effectiveformulation parameters in minimizing alum particle coagulation, thus,maximizing the immunogenicity of the vaccine.

3.5.2 EPI with Powdered Alum-DT

A commercial DT vaccine was used to illustrate the effect of the SFDprocess on an alum phosphate adjuvant-containing vaccine composition.The Alum-DT vaccine was dried by either conventional spray drying (“SD”)or SFD, and the dried powder was then used to immunize hairless guineapigs by using EPI. EPI delivers dry powder directly into the epidermallayer where abundant antigen presenting cells (APCs) can be activated tophagocytose or endocytose the dissolved antigen. The study design of theguinea pig study is shown below in Table 3.4. TABLE 3.4 In vivoimmunogenicity study for Alum-DT powder formulations. Group Powder dT &tT dose/mg (n = 8) Formulation Formation Particle size powder 1 S SD38-53 μm 1.5 Lf/0.5 mg powder 2 T SFD 38-53 μm 1.5 Lf/0.7 mg powder 3Control Liquid N/A 1.5 Lf formulation

The formulations used in the study were as follows. Formulation S: alumphosphate (5 w/v %)/trehalose (5 w/v %). Formulation T: alum phosphate(1.5 w/v %)/trehalose (1.5 w/v %)/glycine (0.4 w/v %)/dextran (0.6 w/v%). Control: the commercial Alum-HBsAg product (Rhein Biotech) used assupplied by the manufacturer.

Formulation S (trehalose-based) was spray-dried using a laboratory-scalespray dryer (Mobile Minor, Niro, Inc). Formulation T, based on thecombination of trehalose, glycine, and dextran, was produced using theSFD method of the present invention. For both powder formulations(Formulations S and T), the dried powder was subjected to a C/G/Stechnique (sieved to 38-53 μm size fraction) in order to match the sizeof the SD powders. A dose of 1.5 Lf for both dT and tT was used, whichis equivalent to approximately 0.5-mg of the SD powders and 0.7-mg ofthe SFD powders based on total protein analysis.

Further evaluation of alum coagulation by optical microscopy andparticle size analysis revealed the same findings that alum particleswere highly coagulated in the SD powder whereas the SFD powders yieldedmore gel-like suspensions upon rehydration. Serum samples were collected4 weeks after prime and two weeks post boost. Serum antibodies weredetermined using the standard anti-dT and tT ELISA. The results aresummarized in FIG. 14 a (anti-dT response) and 14b (anti-tT response).It is apparent that the SD formulation elicited either no (for dT, seeFIG. 14 a) or weak (for tT, see FIG. 14 b) antibody responses. Incontrast, however, the SFD formulation elicited significantly higherresponses that were substantially equivalent to the responses induced bythe untreated liquid vaccine (Control) that was injectedintramuscularly.

Example 4 Optimization of Alum-Adjuvanted Vaccine Compositions Preparedby SFD

4.1 Objectives:

-   -   To optimize the performance of SFD alum-adjuvanted vaccine        powders and enhance the safety profile of the product by        reducing alum content in the final composition, and to        particularly address the following issues: (a) further reduction        of gel coagulation; (b) reduction in local tolerability issues        associated with alum adjuvants; and (c) increase in vivo potency        of the vaccine composition when administered by EPI.        4.2 Materials:

The chemicals and excipients that were used in this study are summarizedbelow in Table 4.1. All alum formulations were concentrated to a desiredconcentration by centrifugation (Allergra 6R centrifuge, BeckmanInstrument, Palo Alto, Calif.) prior to use. TABLE 4.1Chemicals/excipients used in the study. Chemical Lot # Source CommentAluminum phosphate 8934 Accurate Chemical and Manufactured (Adjus-Phos,2% Scientific (Westbury, NJ) by HCI AlPO₄) Biosector (Frederikssund,Denmark) Aluminum hydroxide Accurate Chemical and Manufactured(Alhydrogel, 3% Scientific by Superflos Al(OH)₃) Biosector (Vedbaek,Denmark) Diphtheria toxoid G9334 Accurate Chemical and Manufactured (dT,MW 58 kDa) Scientific by Statens Serum Institute, Denmark, and providedat 5 mg/mL (1 Lf = 2.42 μg), used as supplied Alum phosphate- CSLLimited (Parkville, Bulk adjuvanted DT Australia) containing 5 w/v %alum phosphate adsorbed with both dT and tT at 563 Lf/mL Alum hydroxide-Rhein Amaericana S.A. 20 μg HBsAg adjuvanted hepatitis- (Buenos Ares,Argentina) adsorbed to 0.5 mg B surface antigen of (HBsAg) aluminum or1.5-mg of aluminum hydroxide Dextran (MW 10,000 Da) 18H0568 Sigma (St.Louis, MO) Reagent grade Glycine 28H0103 Sigma Reagent grade Sodiumlauryl sulfate 17H0459 Sigma Sodium dodecyl sulfate (SDS); MW = 288Dalton Mannitol 127H0960 Sigma Reagent grade Pluronic F68 MPCS612B BASF(Mount Olive, NJ) Poloxamer 188NF; MW = 9,000 Trehalose dihydrate28H3797 Sigma Reagent grade Triton X-100 67H0044 Sigma t-Octylphenoxypolyethyethanol; MW = 625 Polysorbate 80 18H5229 SigmaPolyoxyethylenesorbitan monooleate; MW = 1,3104.3 Methods:

In general, placebo aluminum gels were formulated with a variety ofpharmaceutical excipients and dehydrated by SFD. After drying, the drypowder was reconstituted and examined under optical microscopy tomonitor gel coagulation. The degree of coagulation was also determinedby the sedimentation rate of the gel. An optimized formulation processwas then applied to prepare alum phosphate-adjuvanted hepatitis-Bsurface antigen (AlPO₄-HBsAg) where the alum content was 1/20 of thecommercial product. The amount of the antigen was measured in vitro bySDS-PAGE or a micro BCA protein assay, and in vivo potency of theantigen was determined by intramuscular needle/syringe injection and EPIof mice. The local tolerability of SFD alum adsorbed diphtheria-tetanustoxoids (DT) vaccine administered by EPI was assessed in pigs andcompared to intradermal (ID) injection.

4.3.1 Spray-Freezing-Drying (SFD)

The liquid formulation was delivered by the peristaltic pump (Model#77120-70, MasterFlex C/L, Barnant Company, Barrington, Ill.) at a flowrate of 2.0 mL/min into the ultrasonic atomizing system (Sono-TekCorporation, Milton, N.Y.) which consists of a spraying nozzle (Model#05793) and a power supply (Model #06-05108). The nozzle is equippedwith a quasi-electric quartz crystal capable of vibrating at a specificfrequency that determines the size of the droplets. The frequency of 60kHz spraying nozzle produces droplets mostly within the range of 20-80μm. Atomized droplets were sprayed into the liquid N₂-containing pan(16-cm in diameter by 6-cm in height). For formulations subjected to thespray-freezing/thawing experiment, the frozen powder was transferred toa glass vial and thawed under ambient conditions. For frozen dropletsundergoing drying, the pan containing frozen particles in liquidnitrogen was transferred to a pre-cooled (−55° C.) shelf freeze dryer(Model #TDS2C2B5200; Dura-Stop, FTS System, Stone Ridge, N.Y.). Liquidnitrogen evaporated in a few minutes. The freeze-drying condition wasset at −25° C. for 18 hours and 20° C. for 10 hours. The ramping ratewas 1° C./minute consistently. The vacuum pressure was 100 mT throughoutthe cycle. At the end of drying, the powder-containing pans weretransferred into a dry box purged with nitrogen (at <30% relativehumidity) for powder collection. The same lyophilization cycle was usedfor freeze drying the liquid formulations as the freeze-dried samples.

4.3.2 Powder Formation by C/G/S Technique

To prepare powders of high density for EPI, some SFD formulations werecompressed in a stainless steel dye of 13-mm in diameter (Carver Press,Wabash, Ind.) at a pressure of 12,000-15,000 pounds for 5-10 minutes.The compressed discs were ground manually using a mortar and pestle, andthen the ground powder was manually sieved into the size fraction of53-75 μm using 3-inch sieves (Fisher Scientific Products, Pittsburgh,Pa.).

4.3.3 Optical Microscopy

Visual analysis of the particles was performed using an opticalmicroscope (Model DMR, Leica, Germany) with 10×-eyepiece lens and10×-objective lens. The system was equipped with a Polaroid camerasystem for image output.

4.3.4 Scanning Electron Microscopy

The external morphology of particles was examined using an Amray 1810Tscanning electron microscope (Amray, Bedford, Mass.). The powder samplewas first sputtered coated with gold using a Hummer JR Technics unit(Pergamon Corporation, King of Prussia, Pa.).

4.3.5 Particle Size Analysis

The mean geometric/aerodynamic diameter of particles in the volumedistribution was determined using a time-of-flight particle sizeanalyzer (Aerosizer, API, Minneapolis, Minn.). The mean volumetric sizewas calculated using the equipment software and applying a density of1.0 and such that the particle population between 10% (D₁₀) and 90%(D₉₀) could be determined for particle size distribution. Each analysisrequired approximately 3-5 mg of the powder sample.

4.3.6 X-Ray Powder Diffraction (XRD)

XRD measurement was conducted using a 35 kV×15 mA Rigaku (D/max-β, CuKαradiation) X-ray diffractometer at room temperature and ambienthumidity. Samples were scanned at 0.1 degrees/second with 1 second counttime per increment. The range scanned was from 5 to 40 degrees.

4.3.7 Alum Gel Coagulation Analysis

Two methods were used to determine alum gel coagulation, opticalmicroscopy and gel sedimentation. The powder sample was firstreconstituted in water to a concentration of 100 mg/mL withoutagitation. The gel solution was pipetted on a glass slide and examinedunder an optical microscope (Model DMR, Leica, Germany). The same gelsolution was then loaded in a 15-mL polystyrene conical tube (Falcon,Becton Dickinson, Franklin Lakes, N.J.) and the sedimentation rate ofthe alum gel was monitored.

4.3.8 HBsAg Adsorption to Alum Phosphate

To prepare 500 vaccine doses where each dose contains 20 μg HBsAgadsorbed onto 25 μg AlPO₄ in 100 μL, 8.9 mL of HBsAg antigen (1.4 mg/mL,pH 5.5) was mixed with 2.83 mL of AlPO₄ adjuvant (Adju-Phos, 2% AlPO₄),25 mL of 1.8% NaCl, and 13.3 mL of double-distilled water. The mixturewas inverted 5 times and then gently stirred overnight at roomtemperature (RT). The pH was then raised to 7.2 and the mixture wascentrifuged at 4,500 rpm for 20 min at RT. The supernatant was decantedand the pellet was re-suspended in 0.9% normal saline solution. Theamount of protein in the supernatant was determined by BCA proteinassay.

4.3.9 SDS-PAGE

Coomassie colloidal-stained SDS-polyacrylamide gel electrophoresis(SDS-PAGE) was performed on a Nu-PAGE gel from Novex (San Diego, Calif.)(4-12% MES, running buffer, sample buffer, and/or Dithiothreitolreducing agent). The alum-adjuvanted powder formulations wasreconstituted in water and centrifuged to remove the supernatant. Thealum pellet was re-suspended in 200 mM sodium phosphate, pH 7 with 0.1%SDS. The liquid suspension was then mixed with sample buffer from theNovex gel kit. The cocktail samples were then heated at 95° C. for 5minutes and vortexed prior to loading on the gel. The gels were run for35 minutes at 200V/120 mA/25W using a power supply (PowerEase 500,Novex), and then coomassie stained (Novex Colloidal Blue Stain) anddestained with water. The gel images were scanned on a gel scanner(Model GS-700 Imaging Densitometer, BioRad) equipped with a quantitationsoftware (Quantity One), which can quantify the intensity of the gelbands. The unit of signal intensity is Optical Density (O.D.). Allsamples were compared against a molecular weight marker (Mark 12,Novex).

4.3.10 Immunization and Serum Collection

Hairless guinea pigs (Charles River, Wilmington, Mass.) were used toassess the immunogenicity of powder formulations following epidermalpowder injection as described above in Example 3. In general, the methodof immunization was as follows. One mg of powder was dispensed into atrilaminate cassette. The cassette was inserted into the needlelesssyringe delivery device at the time of immunization. The device wasplaced against the left inguinal skin of the animals and actuated byreleasing the compressed helium at 40-bar pressure from the gascylinder. Control animals were immunized with 0.20 mL of liquid vaccinein saline by intramuscular (IM) injection using a 26½-gauge needle.Blood was collected via the kerotid blood vessel prior to eachvaccination and two weeks post boost.

4.3.11 Local Reactogenicity Test

Pigs were anesthetized with a 1:1 mixture of Rompun and Telazol. Theabdomen of each pig was tattooed on either side of the shot site. Eachsite received 2-mg powder formulation by EPI. Control sites received anintra-dermal (ID) injection of reconstituted powders or unprocessedliquid vaccine. After immunization, each injection site was inspected bypalpation weekly.

Skin biopsies from the immunization sites were excised on day 42, fixedwith 10% formalin, and embedded with paraffin. Sections of 6-μmthickness were cut, stained with hematoxylin and eosin (H&E), andvisualized under a light microscope (Nikon, Melville, N.Y.).

4.3.12 ELISA

The antibody response to HBsAg was determined using a modified ELISAmethod (see, e.g., Chen et al. (2000) Nature Med 6:1187-1190). Inparticular a 96-well plate (Costar, Fisher Scientific Products,Pittsburgh, Pa.) was coated with 0.1 μg of antigen in 30 mM phosphatebuffered saline (PBS), pH 7.4, per well and allowed to sit overnight at4° C. Plates were washed 3 times with tris-buffered saline (TBS), pH7.4, containing 0.1% Brij-35, and incubated with test sera diluted inPBS containing 5% dry milk for 1.5 hour. A standard serum, containing aknown level of antibodies to HBsAg, was added to each plate and used tostandardize the titer in the final data analysis. The plates were thenwashed and incubated with biotin-labeled goat antibodies specific formouse immunoglobulin IgG or IgG subclasses (1:8,000 in PBS, SouthernBiotechnology Associate, Birmingham, Ala.) for 1 hour at roomtemperature. Following three additional washes, plates were incubatedwith streptavidin-horseradish peroxidase conjugates (SouthernBiotechnology) for 1 hour at room temperature. Finally, plates werewashed and developed with TMB substrate (Bio-Rad Laboratories, Melville,N.Y.). The endpoint titers of the sera were determined by 4-parameteranalysis using the Softmax Pro 4.1 program (Molecular Devices,Sunnyvale, Calif.) and defined as the reciprocal of the highest serumdilution with an OD reading above the background by 0.1. A referenceserum with a pre-determined titer was used on every plate to calibratethe titers and adjust assay-to-assay and plate-to-plate variation.

4.4 In Vivo Performance, Enhanced Safety:

The following study was carried out to demonstrate the improved safetyof alum-adjuvanted vaccines prepared using the SFD methods of thepresent invention. In particular, granuloma formation was assessed.Granuloma formation is the most common side effect associated withalum-adjuvanted vaccines administered intradermally or subcutaneously.

4.4.1 Local Reactogenicity

The local reactogenicity to SFD alum-adjuvanted dT vaccine deliveredusing EPI was examined and compared with that of ID injection. Thedomestic white pig was chosen as an animal model for this test becauseit's epidermis is structurally similar to that of the human. Both EPIand ID injection with alum-adsorbed dT caused an erythema response(localized skin reaction). The size of the erythema area was observed tobe larger and more intense for the EPI administrations. In all cases,the erythema completely resolved within 48 hours. The site of the EPIadministrations appeared yellowish for an additional 2-3 days, but thenrestored to its normal color. becoming visually indistinguishable fromnormal skin 7 days post treatment. The study matrix and the results fromthe reactogenicity study are reported below in Table 4.2. TABLE 4.2Granuloma formation following administration of Alum-dT¹ Granuloma sitesout of a total ten sites Formulation Alum Route D7 D14 D21 D28 D35 D42liquid Al(OH)3 ID 10 8 8 7 7 7 liquid AlPO4 ID 10 10 10 10 8 8 Powder A²Al(OH)3 EPI 0 0 0 0 0 0 Powder B² AlPO4 EPI 0 0 0 0 0 0¹Each site was treated with 500 μg of alum-absorbed dT (Alum-dT) by IDinjection of liquid vaccine or EPI of SFD powdered vaccine on day 0.Granuloma formation was initially determined by weekly palpation for 6weeks, and then confirmed by histology on day 42.²Powder A and Powder B were produced by formulating the respectivealuminum salt-adsorbed dT with 50% trehalose followed by a combined SFDand C/G/S process to produce 38-53 μm particles.

As can be seen, EPI administration of powdered compositions containingalum hydroxide or alum phosphate adjuvant did not result in granulomasas determined by visual examination (Table 4.2). In contrast, most ofthe ID injection sites had a solid lump that could be detected bypalpation starting from day 7 and until the end of the study. At the endof the study (42 days post treatment), the sizes of the lumps in theanimals receiving the ID liquid vaccine injections ranged in 0.3 to 0.5mm in diameter and 0.2-0.3 mm in thickness.

The results of a histological examination of the EPI sites 42 days aftertreatment are depicted in FIGS. 15 a-15 e. In particular, histologicalexamination showed a skin structure (see FIG. 15 b) and cellularcomposition (FIG. 15 c) that resembled those of normal skin (see FIG. 15a). The lumps from the ID injection sites were found to be typicalgranulomas, composed of inflammatory cells with a connective tissuecapsule (FIG. 15 d). The inflammatory cells were surrounded by a capsuleof connective tissue that had completely replaced the normal tissue,again characteristic of granuloma formation. A closer examination showedthat the main class of infiltrating cells were macrophages (see FIG. 15e). Both the alum hydroxide and alum phosphate liquid vaccinecompositions induced granulomas when introduced by ID injection. Theseresults demonstrate that EPI administration of the powdered vaccinecompositions of the present invention provides a unique safety advantageover conventional administration routes.

4.5 Optimization of the Powder Formulation:

Formulation parameters affecting the stability of the alum gel wereevaluated and optimized to produce SFD particles with minimal alum gelcoagulation and superior particle characteristics suitable for epidermalpowder injection techniques. In this regard, the SFD process had alreadybeen adjusted to achieve the greatest freeze rate possible by atomizingalum-containing liquid formulation into liquid nitrogen, therebyminimizing large-scale alum coagulation from occurring. In this study,the enhancing effects of various excipient combinations wereinvestigated.

4.5.1 Effect of Excipient Composition

High particle density and appropriate particle size are two importantpowder properties that must be optimized for use in transdermal particleadministrations such as EPI. It was expected that excipient compositionwould have a quantifiable influence on the density of the SFD powder,since voids left behind in the particles by evaporation of ice crystalsmay cave-in during drying. Accordingly, a series of alum hydroxide andalum phosphate compositions stabilized with various weight combinationsof trehalose, mannitol and dextran excipients were prepared and thenassessed for gel performance and particle characteristics. SFD particleswere initially visually assessed using SEM, and the results from thestudy are depicted in FIGS. 16 a and 16 b. In particular, thecombination of trehalose/mannitol/dextran=30%/30%/40% and a total solidscontent of 35 w/w % resulted in particles with a corrugated morphology.SEM demonstrated such morphology for the particles containing aluminiumhydroxide at 36 μg/1-mg powder (FIG. 16 a) and for these containingaluminium phosphate at 50 μg/1-mg powder (FIG. 16 b), where theparticles appear to have shrunk and thus become densified during thedrying process.

Next, alum coagulation was evaluated using optical microscopy onreconstituted powders that had been prepared by either a FD or SFDprocess. The results are depicted in FIGS. 17 a-17 d, wherein FIG. 17 ashows the FD alum hydroxide composition, FIG. 17 c shows the FD alumphosphate composition, FIG. 17 b shows the SFD alum hydroxidecomposition, and FIG. 17 d shows the SFD alum phosphate composition. Ascan be seen, both FD formulations were highly coagulated when comparedto their SFD counterparts. Sedimentation experiments confirmed that theFD suspensions had settled, i.e., the top solution became clear, in oneminute but the SFD solutions took approximately 5 hours to clear. Whenthe composition of trehalose/mannitol/dextran excipient was varied, thedensity of the corresponding SFD powder also changed. However, thedifference in coagulation was not distinguishable under opticalmicroscopy. Interestingly, the reconstituted gel settled at faster ratesas the content of mannitol was decreased. Sedimentation of aluminumphosphate (50 μg/1-mg powder) formulated in trehalose/mannitol/dextranat 40%/20%/40% completed, for example, in approximately 2 hours.

4.4.2 Effect of Surface-Active Agents

The stabilizing effect of four common surface-active agent excipients onthe SFD alum-containing particles of the present invention wereassessed. In particular, polysorbate 80 (786 Å, MW=1310), sodium laurylsulfate (SDS), (53 Å, MW=288), Pluronic F68, a block copolymer ofpolyoxyethylene and polyoxypropylene (MW=9,000), and Triton X-100,octylphenol ethylene oxide condensate, average MW=625 were selected forthe study. All four surfactants were formulated withtrehalose/mannitol/dextran excipients (present at 30%/30%/40%) at 3different concentrations, 0.5, 2, and 5% of the solid. Upon examinationof each reconstituted suspension solution by optical microscopy andsedimentation, no significant differences in alum coagulation among thevarious surface-active agents were observed. However, powders containingthe polysorbate 80, SDS, and Triton X-100 agents showed deterioratedpowder flowability, particularly at higher concentrations.

4.4.3 Critical Concentration of Aluminum Salt

Although alum-adsorbed vaccines of low alum concentrations (<0.1 w/v %)have reportedly been freeze-dried with preserved immunogenicity (see,e.g., U.S. Pat. No. 4,578,270 and European Patent No. 01306.19 B1),these formulations are impractical considering that most commercialalum-adjuvanted vaccines are administered at a much higher alumconcentration, typically about 2-3 w/v %. However, as shown in Example 3above, the specific formulations described in U.S. Pat. No. 4,578,270and European Patent No. 0130619 B1 fail to stabilize alum salt fromcoagulation when freeze-dried at alum salt concentrations higher than1%. Therefore, alum salt concentrations might play a critical role inalum coagulation. Our hypothesis is that alum salt coagulation might beunavoidable when the alum concentration exceeds a critical level,despite the effectiveness of fast freezing by SFD. To identify thiscritical concentration, numerous formulations of aluminum phosphate andhydroxide were prepared by SFD at different alum salt concentrationsbefore SFD, excipient compositions, total solid concentrations, andpayloads. Some of these formulations are summarized below in Table 4.3.TABLE 4.3 Effect of alum concentration of the stability of SFDformulations¹ Total solid Liquid Alum Alum content in content priorConc. Prior to SFD powder to SFD Sample SFD (mg/mL) (μg/mg powder) (w/w%) Coagulation A 26.5 76.0 35 no B 30.3 76.0 40 no C 34.0 114.0 30 yes D28.4 114.0 25 no E 20.1 50.3 40 no F² 11.5 28.7 40 no¹For all samples, alum phosphate was formulated withtrehalose/mannitol/dextran at 30/30/40 wt % containing polysorbate 80 at0.5% of the total solid except Samples E and F that contained PluronicF68 at 0.1%.²Alum hydroxide was formulated at the same composition as otherformulations.

After SFD, the various sample formulations were assessed using opticalmicroscopy to determine degree of alum gel coagulation. Results from thestudy are depicted in FIGS. 18 a-18 d. As a result of the assessment, itwas found that concentration of alum salt in the liquid formulationprior to SFD appears to be the critical factor. In this regard, in therange of 26 to 34 mg/mL of alum salt in the liquid (starting)formulation, there is an obvious transition of alum gel into coagulatedform. Optical microscopy on certain reconstituted powder formulations(the samples reported Table 4.3), revealed the following. Formulation A(26.5 mg/mL) was free from coagulation (see FIG. 18 a). Some coagulatedalum particles were observed in Formulation B at 30.3 mg/mL (see FIG. 18b). However, as the concentration of alum in the starting liquid wasfurther increased to 34.0 mg/mL (Formulation C), alum coagulation becamemuch more pronounced (see FIG. 18 c). Alum coagulation had no directcorrelation with the quantity of alum salt in the powder, asFormulations C and D have the same alum content (114 μg/mg powder) andFormulation D was seen to be coagulation-free (see FIG. 18 d). This isbecause Formulation D was prepared from the liquid suspension of alumsalt at 28.4 mg/mL. Therefore, the critical alum salt concentration forthe SFD process appeared to be 30 mg/mL in the liquid gel. A possibleexplanation for this is that the high-concentration alum gel forms adense matrix and affects the pattern of ice formation, where icecrystals are less likely to push freeze concentrate into smallcompartments compared to the case where the alum gel concentration islower. In addition, alum coagulation showed no correlation with thetotal solid content in the liquid gel prior to SFD (see Table 4.3). Ashigher total solid contents lead to more dense SFD particles, a totalsolid content of 35% or higher is preferred for producing powders foruse in EPI.

4.6 In Vivo Performance of the SFD Vaccine Compositions:

Based upon the above optimization assessment, it was decided that theSFD formulation should optimally start with a liquid formulationcontaining at least about 30 mg/mL alum gel concentration and have a 35%total solid content. SFD of one human dose of the commercial alumadsorbed HBsAg vaccine, which contains 500 μg of alum adjuvant, willyield approximately 28 mg of dried powder. Thus, to reduce the dry mass,the amount of alum in the vaccine dose must be reduced. As indicated bythe tolerability studies also described herein above, reduction in thealum in the vaccine should provide the added benefit of improving thetolerability of the SFD vaccine compositions. Accordingly, SFD powdervaccine compositions were made using 20 μg of the HBsAg antigen adsorbedto 25 μg alum phosphate adjuvant, and the immunogenicity of thisformulation was determined using either EPI or IM injection (afterreconstitution) of the SFD vaccine compositions and compared against thecommercial vaccine that contains the same HBsAg dose, but 20 times morealum adjuvant.

4.6.1 Immunogenicity Studies

We first compared in hairless guinea pigs the immunogenicity of powderedformulations of HBsAg adjuvanted with either alum phosphate or alumhydroxide at a HBsAg:alum ratio of 1:25), denoted as Formulations E andF (Table 4.3), respectively. These formulations were administered byeither EPI or IM injection of a liquid after the SFD particles werereconstituted. The results of the study are depicted in FIG. 19 a. Ascan be seen, all groups showed a significant increase in the antibodyresponse after the first and boost vaccinations (6 and 9 weeks afterprime respectively) were administered (see FIG. 19 a). The antibodytiters in the animals vaccinated with the reconstituted powders wereequivalent to that induced by the untreated liquid formulation vaccine(P>0.05), suggesting the suitability of SFD for stabilization of dryalum formulation. All animals vaccinated by EPI developed significantantibody titers. Interestingly, the two alum salts appear to be equallyimmunogenic when delivered as dry powders in the skin.

In a subsequent study, we evaluated the immunogenicity of the dry powderformulation containing AlPO₄ at 1/20 of the regular alum content, i.e.20 μg of HBsAg adsorbed to 25 μg of aluminium. Because this high dose ofvaccine can overwhelm the immune response in guinea pigs, the subjectswere vaccinated with only 1/10 of the dose (2.0 μg HBsAg/2.5 μg ofALPO₄). The results of the study are depicted in FIG. 19 b, where it canbe seen that the SFD formulation was efficient in eliciting antibodyresponses by IM injection as a reconstituted powder. At 2 weekspost-boost, the highest antibody titers were detected in animals thatreceived the SFD-reconstituted formulation with a geometric mean titer(GMT) of 5.6 (log 10). These titers were similar to those detected inanimals that received the untreated liquid vaccine formulation (GMT=5.5log 10) which contained twenty times more alum in the form of alumhydroxide, suggesting that alum dose in the commercial vaccine can bereduced without compromising the efficacy. This significantly reducedalum dose adds additional safety features to alum-adjuvanted vaccinesdelivered by EPI.

Accordingly, novel spray freeze-dried powder compositions and methodsfor producing these compositions have been described. Although preferredembodiments of the subject invention have been described in some detail,it is understood that obvious variations can be made without departingfrom the spirit and the scope of the invention as defined by theappended claims.

1. A process for the preparation of a powder, which process comprises the step of spray freeze-drying an aqueous solution or suspension comprising a pharmaceutical agent, said solution or suspension having a solids content of 20% by weight or more.
 2. A process according to claim 1, wherein the solution or suspension has a solids content of 30% by weight or more.
 3. A process according to claim 2, wherein the solids content is 40% by weight or more.
 4. A process according to claim 1, wherein the pharmaceutical composition is an antigen.
 5. A process according to claim 4, wherein the antigen is adsorbed in an aluminum salt or calcium salt adjuvant.
 6. A process according to claim 4, wherein the antigen is a bacterial or viral antigen.
 7. A process according to claim 1, wherein the solution or suspension further comprises (a) an amorphous excipient selected from the group consisting of monosaccharides, disaccharides, oligosaccharides and polysaccharides; and (b) a crystalline excipient selected from the group consisting of carbohydrates, sugars and sugar alcohols.
 8. A process according to claim 1, wherein the solution or suspension further comprises (a) an amorphous excipient selected from the group consisting of dextrose, sucrose, lactose, trehalose, cellobiose, raffinose, isomaltose and cyclodextrins, and (b) mannitol as a crystalline excipient.
 9. A process according to claim 7, wherein the solution or suspension further comprises (c) a polymer.
 10. A process according to claim 9, wherein the polymer is dextran.
 11. A process according to claim 7, wherein the solution or suspension further comprises (d) an amino acid or a physiologically acceptable salt thereof.
 12. A process according to claim 7, wherein the solution or suspension further comprises (c) a polymer and (d) an amino acid or physiologically acceptable salt thereof.
 13. A process according to claim 1, wherein the solution or suspension further comprises trehalose, mannitol and dextran in a weight ratio of from about 3:3:4 to about 4:4:3.
 14. A process according to claim 1, wherein the solution or suspension is sprayed from an ultrasonic nozzle.
 15. A process according to claim 1, wherein the solution or suspension is sprayed into liquid nitrogen.
 16. A process according to claim 1, wherein the solution or suspension is sprayed into a liquified gas and the liquified gas containing the resulting frozen droplets of the solution or suspension is subjected to a two stage drying process comprising: (i) a first drying stage which is performed at a temperature of from about −50° C. to 0° C. for a period of about 4 to 24 hours under a pressure of about 20 to 500 mT; and (ii) a second drying stage which is performed at a temperature of from about 5 to 30° C. for a period of about 5 to 24 hours under a pressure of less than 100 mT.
 17. A process according to claim 1, wherein the resulting spray freeze-dried particles are collected, washed and dried.
 18. A process according to claim 17, wherein the dried particles are sieved. 